Acenaphthyl substituted guanidines and methods of use thereof

ABSTRACT

Modulators of neurotransmitter release including substituted guanidines, and N&#39;&#39;-aminoguanidines, and N,N&#39;N&#39;&#39;, N&#39;&#39;&#39;-tetrasubstituted hydrazinedicarboximidamides, and pharmaceutical compositions thereof are disclosed. Also disclosed are methods involving the use of such neurotransmitter release modulators for the treatment or prevention of pathophysiologic conditions characterized by the release of excessive or inappropriate levels of neurotransmitters. Also disclosed are screening assays for compounds which selectively inhibit glutamate release. Also disclosed are methods of blocking voltage sensitive sodium and calcium channels in mammalian nerve cells.

The present invention was made with U.S. government support. The U.S.government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/343,829 filed on Nov.22, 1994, which is a divisional of application Ser. No. 07/833,421,filed Feb. 10, 1992, now U.S. Pat. No. 5,403,861, which is acontinuation of application Ser. No. 07/652,104, filed Feb. 8, 1991, nowabandoned.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry. In particular,this invention relates to modulators of neurotransmitter re lease, andto pharmaceutical compositions comprising the same which possessneuroprotective and other therapeutic utilities. The invention alsorelates to screening assays for compounds which inhibit neurotransmitterrelease. This invention further relates to methods involving the use ofsuch neurotransmitter release modulators for the treatment or preventionof certain pathophysiologic conditions characterized by the excessive orinappropriate release of neurotransmitters.

BACKGROUND OF THE INVENTION

A wide variety of substituted guanidines are disclosed in the patentliterature. For example:

U.S. Pat. Nos. 1,411,731 and 1,422,506 discloses diphenylguanidine as arubber accelerator;

U.S. Pat. No. 1,597,233 discloses N-o-tolyl-N'-phenyl-guanidine as arubber accelerator;

U.S. Pat. No. 1,672,431 discloses N,N'-di-o-methoxyphenylguanidine asbeing useful for therapeutic purposes, especially in the form ofwater-soluble salts;

U.S. Pat. No. 1,730,338 disclosesN-p-dimethyl-amino-phenyl-N'-phenylguanidine as a rubber accelerator;

U.S. Pat. No. 1,795,738 discloses a process for the production ofN,N'-dialkyl-di-substituted guanidines, includingN-di-ethyl-N'-phenyl-guanidine, N-diethyl-N-isoamylguanidine,N-dimethyl-N'-isoamylguanidine and N-dimethyl-N'-ethylguanidine;

U.S. Pat. No. 1,850,682 discloses a process for the preparation ofdisubstituted guanidine rubber accelerators bearing an additionalsubstituent on the imine nitrogen atom;

U.S. Pat. No. 2,145,214 discloses the use of disubstituted guanidines,e.g., diarylguanidines especially dixylylguanidine, as parasiticides;

U.S. Pat. No. 2,254,009 discloses sym-di-2-octyl-guanidine and U.S. Pat.Nos. 2,274,476 and 2,289,542 disclose symdicyclohexylguanidine asinsecticides and moth larvae repellents;

U.S. Pat. No. 2,633,474 discloses 1,3-bis(o-ethylphenyl)guanidine and1,3-bis(p-ethylphenyl)guanidine as rubber accelerators;

U.S. Pat. No. 3,117,994 discloses N,N',N"-trisubstituted guanidines andtheir salts as bacteriostatic compounds;

U.S. Pat. No. 3,140,231 discloses N-methyl- andN-ethyl-N'-octylguanidines and their salts as antihypertensive agents;

U.S. Pat. No. 3,248,246 describes (Example 5) a 1,3-disubstitutedguanidine whose substituents are hydrophobic hydrocarbon groups, one ofwhich is naphthylmethyl and the other is n-butyl;

U.S. Pat. No. 3,252,816 discloses various N-substituted andunsubstituted cinnamyl-guanidines and generically the corresponding N'-and N"-alkyl substituted compounds and their salts as antihypertensiveagents;

U.S. Pat. No. 3,270,054 discloses N-2-adamant-1-yl- andN-2-homoadamant-1-yl-oxy-ethyl-thioethyl- and aminoethyl-guanidinederivatives bearing at most two lower alkyl groups on the N'- and/orN"-nitrogen atom as sympathicolytic and anti-viral agents;

U.S. Pat. No. 3,301,755 discloses N-ethylenicallyunsubstituted-alkyl-guanidines and the corresponding N'- and/or N"-loweralkyl compounds as hypoglycemic and antihypertensive agents;

U.S. Pat. No. 3,409,669 disclosesN-cyclohexylamino-(3,3-dialkyl-substituted-propyl)-guanidines and thecorresponding N'-alkyl- and/or N"-alkyl-substituted compounds ashypotensive agents;

U.S. Pat. No. 3,547,951 discloses 1,3-dioxolan-4-yl-alkyl-substitutedguanidines which have anti-hypertensive activity and discloses loweralkyl, including n-butyl, as a possible substituent on the other aminogroup;

U.S. Pat. No. 3,639,477 discloses propoxylguanidine compounds as havinganorectic properties;

U.S. Pat. Nos. 3,681,459; 3,769,427; 3,803,324; 3,908,013; 3,976,787;and 4,014,934 disclose aromatic substituted guanidine derivativeswherein the phenyl ring can contain hydroxy and/or halogen substituentsfor use in vasoconstrictive therapy;

U.S. Pat. No. 3,804,898 discloses N-benzylcyclobutenyl andN-benzylcyclobutenyl-alkyl-guanidines and the corresponding N-alkyland/or N"-alkyl-substituted compounds as hypotensive agents;

U.S. Pat. No. 3,968,243 discloses N-aralkyl substituted guanidines andthe corresponding N'-alkyl-N"-alkyl and N',N'-aralkyl compounds as beinguseful in the treatment of cardiac arrhythmias;

U.S. Pat. No. 3,795,533 discloses o-halo-benzylidene-aminoguanidines andtheir use as anti-depressants for overcoming psychic depression;

U.S. Pat. No. 4,007,181 discloses various N,N'-disubstituted guanidinessubstituted on the imine nitrogen atom by a adamantyl as possessingantiarrhythmic and diuretic activities;

U.S. Pat. No. 4,051,256 discloses N-phenyl- and N-pyridyl-N'-adamantyland cycloalkyl-guanidines as antiviral agents;

U.S. Pat. No. 4,109,014 discloses N-hydroxysubstituted guanidines andthe corresponding N-methyl disubstituted guanidines as vasoconstrictoragents;

U.S. Pat. No. 4,169,154 discloses the use of guanidines in the treatmentof depression;

U.S. Pat. No. 4,393,007 discloses N-substituted and unsubstituted,N-substituted methyl-N'-unsubstituted, monosubstituted anddisubstituted-N"-unsubstituted and substituted guanidines as ganglionicblocking agents; and

U.S. Pat. No. 4,471,137 discloses N,N,N'N"-tetraalkyl guanidines asbeing sterically hindered bases useful in chemical synthesis.

U.S. Pat. No. 4,709,094 discloses 1,3-disubstituted-guanidines, e.g.,1-3-dibutyl-guanidine and 1,3 di-o-tolyl-guanidine (DTG), as sigma brainreceptor ligands.

For examples of other substituted guanidines, see, e.g., U.S. Pat. Nos.1,422,506; 1,642,180; 1,756,315; 3,159,676; 3,228,975; 3,248,426;3,283,003; 3,320,229; 3,479,437; 3,547,951; 3,639,477; 3,784,643;3,949,089; 3,975,533; 4,060,640 and 4,161,541.

Geluk, H. W., et al., J. Med. Chem. 12:712 (1969) describe the synthesisof a variety of adamantyl disubstituted guanidines as possible antiviralagents, including N,N'-di-(adamantan-1-yl)-guanidine hydrochloride,N-(adamantan-1-yl)-N'-cyclohexyl-guanidine hydrochloride andN-(adamantan-1-yl)-N'-benzylguanidine hydrochloride.

PCT Application Publication No. WO88/00583 disclosesdiadamantylguanidine and N,N'-di-(2-adamantyl)guanidine. These compoundsare reportedly useful for treating schizophrenia, psychosis, anddepression.

Vasilev, P., et al., Chem. Abstr. 93:150095u, discloses a compound withthe following formula: ##STR1##

This compound reportedly has virucidal activity.

Ginsburg, V. A., et al., Chem. Abstr. 4518d (1962), and Ginsburg, V. A.,et al., Zhurnal Organ. Khimii 7:2267-2270 (1971), disclose a compoundhaving the formula: ##STR2##

Kreutzberger and Schuker, Arch Pharmz. Ber. Deut. Pharm. Ges.305:400-405 (1975), disclose a compound having the formula: ##STR3##

German Patent No. DE 2,452,691, discloses a compound having the formula:##STR4## wherein, inter alia, R₀ is hydrogen, halogen, hydroxy, alkoxyor C₁ -C₆ alkyl;

R₁ and R₂ are halogen, hydroxy, C₁ -C₄ alkyl or C₁ -C₄ alkoxy;

R₃, R₃ ' and R₃ " are hydrogen or C₁ -C₄ alkyl;

R₄ is hydrogen, C₁ -C₄ alkyl, hydroxy, C₁ -C₄ alkoxy or an amino group;

R₅ and R₆ are hydrogen, C₁ -C₆ alkyl, or an acyl group, or form a cyclicring. Particular compounds disclosed in this patent document include1-(2,6-dichlorophenyl)-2-(2,6-dichlorobenzylidine)aminoguanidine.HCl and1-(2,6-dichlorophenyl)-2-cyclohexylidene-aminoguanidine. These compoundsreportedly have antihypertensive properties.

Sunderdiek, R., et al., Chem. Abstr. 81:91439m (1974), disclose acompound having the formula: ##STR5## wherein R=Ph or cyclohexyl.

Bent, K. J., et al., Chem. Abstr. 74:63479m (1971), is an abstract ofGerman Patent No. 2,029,707. This patent discloses antiviral compoundshaving the formula: ##STR6## wherein: R is amino, methyl, iso-butyl orp-substituted phenyls;

R¹ is hydrogen, methyl or iso-butyl;

NRR₁ is morpholino;

R₂ and R₃ are hydrogen, CHC₆ H₃ Cl₂ (2,4-), isopropyl,

C₆ H₄ p--Cl, CH(CH₂)₁₀ CH₃ or 1-(4-pyridyl)ethylidene.

Huisgen et al., Chem. Abstr. 63:2975d (1965), disclose a compound havingthe formula: ##STR7##

Kroeger, F., et al., Chem. Abstr. 60:9264f, disclose a compounds havingthe formulae: ##STR8##

Heinisch, L., J. Pract. Chem. 329:290-300 (1987), discloses a compoundhaving the formula: ##STR9##

Kramer, C.-R., et al., Biochem. Physiol. Pflanzen. 178:469-477 (1983),disclose a compound having the formula: ##STR10## wherein R is hydrogen,methyl, butyl, hexyl, benzyl and phenyl, and R' is hydrogen or methyl.These compounds reportedly have algicidic activity.

Prasad, R. N., et al., Can. J. Chem. 45:2247-2252 (1967), disclose acompound having the formula: ##STR11## wherein R and R' are hydrogen orCl₂ --CH--CO--, R₂ is hydrogen, and R' is any one of a number ofsubstituents having the general formula alkyl═N--. These compounds wereevaluated for their antibacterial activity.

Huisgen et al., Chem. Ber. 98:1476-1486 (1965), disclose a compoundhaving the formula: ##STR12##

Podrebarac, E. G., et al., J. Med. Chem. :283-288 (1963), disclosecompounds having the formula: ##STR13## wherein the R groups may behydrogen or methyl. These compounds were intermediates for thepreparation of methylglyoxal bis(guanylhydrazone) analogs which may haveactivity against adult acute myelocytic leukemia.

Kroger et al., Ber. 97:396-404 (1964), disclose a compound having theformula: ##STR14##

Durant, G. J., et al., J. Med. Chem. :22-27 (1966), disclose a compoundhaving the formula: ##STR15## wherein R is an alkyl, halo, or alkoxygroup. These compounds reportedly have antiinflammatory activity.

The amino acid L-glutamate is widely thought to act as a chemicaltransmitter substance at excitatory synapses within the central nervoussystem. Neuronal responses to glutamate are complex and appear to bemediated by at least three different receptor types, i.e., KA, QA andNMDA subtypes, each being named for their relatively specific ligands,i.e., kainic acid, quisaqualic acid and N-methyl-D-aspartic acid,respectively. An amino acid which activates one or more of thesereceptor types is referred to as an excitatory amino acid (EAA).

The NMDA subtype of excitatory amino acid receptors is activated duringnormal excitatory synaptic transmission in the brain. Activation of NMDAreceptors under normal conditions is responsible for the phenomena oflong-term potentiation, a memory-like phenomenon, at excitatorysynapses. Excessive excitation of neurons occurs in epileptic seizuresand it has been shown that over-activation of NMDA receptors contributesto the pathophysiology of epilepsy.

NMDA receptors are also strongly involved in nerve cell death whichoccurs following brain or spinal chord ischemia. Upon the occurrence ofischemic brain insults such as stroke, heart attack or traumatic braininjury, an excessive release of endogenous glutamate occurs, resultingin the over-stimulation of NMDA receptors. Associated with the NMDAreceptor is an ion channel. The recognition site, i.e., the NMDAreceptor, is external to the ion channel. When glutamate interacts withthe NMDA receptor, it causes the ion channel to open, thereby permittinga flow of cations across the cell membrane, e.g., Ca²⁺ and Na⁺ into thecell and K⁺ out of the cell. It is believed that this flux of ions,especially the influx of Ca²⁺ ions, caused by the interaction ofglutamate with the NMDA receptor, plays an important role in nerve celldeath. See, e.g., Rothman, S. M. and Olney, J. W., Trends in Neurosci.10(7):299-302 (1987).

In vitro studies have clearly demonstrated that activation of KAreceptors can cause excitatory neuronal damage, although longerexposures are required (Koh, J. Y. et al., J. Neurosci. 10:693-705(1990); and Frandsen, A. J. et al., J. Neurochem. 33:297-299 (1989)).The competitive KA receptor antagonist2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinozaline (NBQX) is effective inpreventing delayed neuronal degeneration following transient forebrainischemia in rodents (Sheardown, M. J. et al., Science 247:571-574(1990). However, such effects require relatively large and potentiallytoxic systemic doses of NBQX, apparently because this compound exhibitspoor penetration of the blood-brain barrier.

At present, there is a critical need for effective treatments whichlimit the extent of nerve cell death following a stroke or traumaticbrain injury. Recent advances in the understanding of the mechanismsunderlying this nerve cell death have led to the hope that a drugtreatment can be developed. Research and development efforts in thisarea have focussed on blocking the actions of glutamate that aremediated by the NMDA receptor-channel complex. Two approaches are welldeveloped: competitive NMDA receptor antagonists (Choi D. W. (1990)Cerebrov. Brain Metab. Rev. 1:165-211; Watkins, J. C. and Olverman, H.J. (1987) Trends Neurosci. 10:265-272) and blockers of the ion channelof the NMDA receptor-channel complex (Meldrum, B. (1990) CerebrovascularBrain Metab. Rev. 2:27-57; Choi, D. W. (1990) Cerebrovascular BrainMetab. Rev. 2:105-147; and Kemp, J. A. et al., Trends Neurosci.10:265-272 (1987)). The ion-channel blocker MK-801 is an effectiveneuroprotective agent in a variety of in vivo models of stroke (Meldrum,B. (1990) Cerebrovascular Brain Metab. Rev. 2:27-57; Albers, G. W. etal., Annals Neurol. 25:398-403 (1989)). However, there is some toxicityassociated with this class of compounds (Olney, J. W. et al., Science244:1360-1362 (1989); Koek, W. and Colpaert, J. (1990) J. Pharmacol.Exp. Ther. 252:349-357) and NMDA antagonists have been shown to inhibitmemory acquisition (Morris, R. G. M. (1988) in Excitat. A.A.'s in Healthand Disease, D. Lodge (ed.), Wiley, 297-320). These side effects maylimit the clinical use of such agents to acute situations.

Blockers of neurotransmitter release have also received some attentionas potential neuroprotective agents. For example, adenosine analogs mayindirectly attenuate neurotransmitter release via G-protein-mediatedinhibition of presynaptic Ca channels (Meldrum, B. Cerebrovascular andBrain Metab. Rev. 2:27-57 (1990); Dolphin, A. C. Nature 316:148-150(1985)). It has been shown that such compounds are neuroprotectiveduring ischemia in various rodent models of stroke (Evans, M. C. et al.Neurosci. Lett. 83:287-292 (1987)). Ault, B. and Wang, C. M., Br. J.Pharmacol. 87:695-703 (1986), disclose that adenosine inhibitsepileptiform activity in hippocampal slices.

Other putative blockers of glutamate release act by an as yet undefinedmechanism. The substitutedpiperidinederivative2-(4-(p-fluorobenzoyl)piperidin-1-yl)-2'-acetonaphthone(E-2001) (Kaneko, T. et al. Arzneim-Forsch./Drug Res. 39:445-450 (1989))and the compound PK 26124 (riluzole,2-amino-6-trifluoromethoxybenzothiazole) (Malgouris, C. et al. J.Neurosci 9:3720-3727 (1989)) have been shown to be neuroprotective inrodents. PK 26124, at therapeutic dosages in rats, does not seem toproduce MK-801-like behavioral side effects in a controlled comparisonof its efficacy/safety ratio with that of MK-801 (Koek, J. W. andColpaert, F. C., J. Pharmacol. Exp. Ther. 252:349-357 (1990)). E-2001ameliorates the degeneration of pyramidal neurons in the hippocampal CA1sector following transient ischemia in Mongolian gerbils. In addition,E-2001 improves stroke symptoms induced by permanent unilateral carotidartery ligation in gerbils, prolonged the survival time followingpermanent bilateral carotid artery ligation in gerbils and mice, andprolonged the survival time following intravenous injection of KCN intomice. Kaneko, T. et al., Arzneim.-Forsch./Drug Res. 39:445-450 (1989).

It is believed that glutamate neurotoxicity is involved in acute injuryto the nervous system as observed with seizure, hypoxia, hypoglycemia,and trauma, as well as in chronic degenerative diseases such asHuntington's disease, olivopontocereballar atrophy associated withglutamate dehydrogenase deficiency and decreased glutamate catabolism,Guam amyotrophic lateral sclerosis/Parkinsonium-dementia, Parkinson'sdisease, and Alzheimer's disease. Choi, D. W., Neuron 1:623-634 (1988);Choi, D. W., Cereb. Brain Met. Rev. 2:105-147 (1990).

Langlais, P. L. et al., J. Neurosci 10:1664-1674 (1990), disclose thatglutamate and GABA may be involved in pyrithiamine-induced thiaminedeficiency (PTD) which causes the formation of thalamic lesions andseizures. Administration of the NMDA receptor antagonist MK-801 duringthe late stages of PTD resulted in a marked attenuation of necroticdamage to the thalamus and periacqueductal gray and a reduction in thenumber and size of hemorrhagic lesions. Since thiamine deficiency isresponsible for similar damage in Wernicke-Korsakoff's syndrome,Langlais et al. suggest that these results provide an important rationalfor the treatment of this human neuropathic condition.

Malgouris, C. et al., J. Neurosci. 9:3720-3727 (1989), disclose that PK26124 (riluzole), a compound which inhibits glutamate release from nerveterminals, has anticonvulsant activity, improves sleep quality inrodents, and is active in protecting the rodent brain from the cellularand functional consequences of ischemia, including the prevention ofmemory loss and hippocampal neuronal damage.

Miller, et al., New Anticonvulsant Drugs, Meldrum, B. S. and Porter R.J. (eds), London:John Libbey, 165-177 (1986), disclose that theglutamate release blocker lamotragine is an anticonvulsant.

Price, M. T. and Olney, J. W., Soc. Neurosci. Abstr. 16:377, abstr.161.16 (1990), disclose that the administration of EAA antagonistscompletely prevented emesis in ferrets that were subject to chemotherapywith cisplatin. The EAA antagonists employed did not penetrate theblood-brain barrier, and it was thus suggested that such compounds wayprevent nausea, a common side effect during cancer chemotherapy.

Calcium antagonists such as nimodipine act both as cerebral vasodilators(Wong, M. C. W. and Haley, E. C. Jr., Stroke 24:31-36 (1989)), and toblock calcium entry into neurons (Scriabine, A. Adv. Neurosurg. (1990)).Modest improvement in the outcome of stroke has been observed inclinical trials (Gelmers, H. J. et al., N. Eng. J. Med. 318:203-207(1988)). While there are significant cardiovascular side effects,nimodipine appears less toxic than the NMDA antagonists and may find arole in the chronic treatment of stroke and other neurologicaldisorders.

There are at least 3 subclasses of Ca channels, "T", "N", and "L", thatdiffer in their pharmacology, location in neuronal and non-neuronaltissues, and physiological properties (Nowycky, M. C. et al. Nature316:440-443 (1985); Bean, B. P. Ann. Rev. Physiol. 51:367-384 (1989)).Voltage-sensitive calcium channels (VSCC) in presynaptic nerve terminalscontrol the influx of Ca²⁺ and thereby determine the quantity andduration of transmitter released by the presynaptic action potentials.Biochemical ⁴⁵ Ca tracer flux experiments with isolated nerve endings(synaptosomes) indicate that K⁺ -depolarization dependent ⁴⁵ Ca entryconsists of fast transient and slow sustained components. The transientCa influx has been determined to represent a channel mediated process,whereas the sustained component reflects Ca entry via reversed Na/Caexchange (Turner, T. and Goldin, S., J. Neurosci. 5:841-849 (1985);Suskziw, J. B. NATO ASI Series, H21:286-291 (1988); Suszkiw, J. B. etal. J. Neurochem. 42:1260-1269 (1989)).

European Patent Application No. 0 266 574, discloses that calciumoverload blockers will be useful in the treatment of anoxia, ischemia,migraine and epilepsy. This application also discloses that certainpiperidine derivatives have activity against calcium overload in thebrain and may be used in the treatment of migraine.

Dreyer, E. B. et al., Science 248:364-367 (1990), disclose that theHIV-1 coat protein gp120 produces neuronal cell injury which may beresponsible for the dementia and blindness encountered in acquiredimmunodeficiency syndrome (AIDS). Calcium channel antagonists preventedthe gp120-induced neuronal injury of retinal ganglion cells. Dreyer etal. propose that calcium channel antagonists may prove useful inmitigating HIV-1 related neuronal injury.

SUMMARY OF THE INVENTION

It is an object of this invention to provide substituted guanidines,amino guanidines and N,N',N"N'"-tetrasubstitutedhydrazinedicarboximidamides which modulate the release ofneurotransmitters (e.g., glutamate) from neuronal cells.

Some disorders (e.g., neuronal damage in stroke) may be alleviated byinhibiting the release of EAAs such as glutamate. Some disorders (e.g.,depression) may be alleviated by inhibiting the release of inhibitoryneurotransmitters such as gammaaminobutyric acid (GABA). Furthermore,inhibiting the release of an excitatory neurotransmitter (glutamate) mayindirectly potentiate the release or the subsequent actions of aninhibitory transmitter (e.g., GABA), and thus serve to treat disordersknown to be alleviated by more direct potentiation of inhibitoryneurotransmission (in this example, anxiety and/or insomnia). Thisserves to illustrate the broad scope of the therapeutic potential of thecompounds of the present invention. Thus, any disease that results frommodulation of a particular neurotransmitter system can be counteractedby the substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention which act either on the same or another class ofneurotransmitters.

It is yet a further object of the invention to treat or prevent nervecell death resulting from hypoxia, hypoglycemia, brain or spinal cordischemia, brain or spinal chord trauma, stroke, heart attack, ordrowning, by the administration of effective amounts of substitutedguanidines, amino guanidines and N,N', N",N'"-tetrasubstitutedhydrazinedicarboximidamides which inhibit the release ofneurotransmitters from neuronal cells.

It is yet a further object of the present invention to treat or preventvarious neurodegenerative diseases such as Huntington's disease,Amyotrophic Lateral Sclerosis, Alzheimer's disease, Down's Syndrome,Korsakoff's disease, olivopontocerebellar atrophy, HIV-induced dementiaand blindness or multi-infarct dementia, by the administration ofeffective amounts of substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides which inhibitthe release of neurotransmitters.

A further object of the invention is to provide substituted guanidines,amino guanidines and N,N',N", N'"-tetrasubstitutedhydrazinedicarboximidamides which are useful for the treatment orprevention of neurological conditions such as epilepsy and convulsions,carbon monoxide poisoning, cyanide poisoning, toxic brain damage causedby, for example, tetrodotoxin or shellfish toxins, anxiety, amnesia,migraine, river blindness, and nausea which may result fromchemotherapy.

It is also an object of the present invention to provide a screeningassay for inhibitors of neurotransmitter release (e.g. glutamate,dopamine, norepinephrine, glycine, aspartate, serotonin, and otherneurotransmitters), from brain nerve terminals. It is also an object ofthe invention to provide a new screening assay which allows for theidentification of new compounds which inhibit certain kineticsubcomponents of glutamate release. Such subcomponents can only beidentified by an assay system capable of resolving neurotransmitterrelease on the subsecond time scale.

The screening assay of the invention comprises the following steps:

(a) contacting immobilized synaptosomes containing radiolabelledneurotransmitter with a compound suspected of inhibitingneurotransmitter release;

(b) inducing, by depolarization, the release of radiolabelledneurotransmitter from the immobilized radiolabelled synaptosomesobtained in step (a);

(c) washing the immobilized radiolabelled synaptosomes obtained in step(b) with a buffer comprising said compound and fractionating theeffluent every 15 to 500 msec; and

(d) detecting the relative amount of radiolabelled neurotransmitter ineach fraction compared to control synaptosomes which have not beenexposed to the compound of interest;

wherein a reduced amount of released radiolabelled neurotransmitter inthe fractions from synaptosomes treated with the compound relative tocontrol synaptosomes indicates that the compound inhibitsneurotransmitter release.

The screening assay may be conducted in the presence or absence of Ca²⁺in order to identify those compounds which are selective inhibitors ofone or more subcomponents or neurotransmitter release.

It is a further object of the present invention to provide novelsubstituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides as well aspharmaceutical compositions thereof.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanying Figureswherein:

FIG. 1 depicts a graph showing ³ H glutamate release from rat brainsynaptosomes in the presence and absence of 2.4 mM Ca²⁺.

FIG. 2 depicts a graph showing the effect of 0 μM, 10 μM, 30 μM, and 100μM N,N'-di-(adamantan-1-yl)guanidine on the Ca²⁺ -dependent ³ Hglutamate release from rat brain synaptosomes.

FIG. 3 depicts a graph showing the effect of 0 uM, 1 uM, 3 uM and 10 uMof N,N'-di(5-acenaphthyl)guanidine over time on the Ca²⁺ -dependentrelease of [³ H]glutamate from radiolabelled synaptosomes after K⁺depolarization (at time 0).

FIG. 4 depicts a graph showing the correlation between inhibition ofsynaptosomal ⁴⁵ Ca uptake by 10 μM ofN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine (#1),N-(1-naphthyl)-N'-(m-ethylphenyl)-N'-methylguanidine (#2),N,N'-di-(1-naphthyl)guanidine (#3), N,N'-di-(adamantan-1-yl)guanidine(#4), N,N'-di-(adamantan-2-yl)guanidine (#5),N,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide (#6) andN,N'-di(5-acenaphthyl)guanidine (#7) and the inhibition of the tonic(persistent) Ca-dependent component of glutamate release, i.e. thecomponent of release which does not rapidly decay, but persists for atleast several seconds following depolarization. FIG. 4 further depictsthe non-correlation between inhibition of synaptosomal ⁴⁵ Ca-uptake by10 μM of the above-mentioned compounds and the inhibition of the phasiccomponent of glutamate release.

FIG. 5 depicts a graph showing the effects of various concentrations ofN,N'-di-(adamantan-2-yl)guanidine on the Ca²⁺ -dependent glutamaterelease stimulated with the sodium channel activator veratridine.

FIG. 6 depicts a graph showing the inhibition of potassium-stimulated Cauptake of synaptosomes by N,N'-di-(adamantan-2-yl)guanidine (▪),N,N'-di-(1-naphthyl)guanidine (♦; .sup....... IC₅₀ =9.1 μM, ----- IC₅₀=16 μM), N,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide (;₋₋.sup..₋₋ IC₅₀ =3.3 μM), and N,N'-di-(adamantan-1-yl)guanidine (Δ; ₋₋IC₅₀ =6.6 μM) against the percent of Ca uptake compared to controlsynaptosomes.

FIG. 7 depicts a graph showing the sodium channel blockade byN,N'-di(5-acenaphthyl)guanidine in N1E-115 cells, compared to controlcells not treated with the drug. The stimulus protocol is shown on thetop of the figure.

FIG. 8 depicts a graph showing the Ca-independent and Ca-dependentrecovery of ³ H-glutamate release from synaptosomes over time.

FIG. 9 depicts a graph showing the Ca-independent and Ca-dependentrecovery of ³ H-glutamate release from synaptosomes, as a percentage ofthe first pulse.

FIG. 10 depicts a graph showing an electrophysiology study on N-typechannels of single bullfrog dorsal root ganglion cells treated with 5 uMof N,N'-di(acenaphthyl)guanidine.

FIG. 11 depicts a hippocampal slice preparation containing a stimulatingand recording electrode.

FIG. 12A depicts a graph showing the results from the bath applicationof N,N'-di(5-acenaphthyl)guanidine on population spikes and field EPSP.

FIG. 12B depicts a graph showing the results from the bath applicationof N,N'-di(5-acenaphthyl)guanidine on the field EPSP after electricalstimulation.

FIG. 12C depicts the effect of 20 uM of N,N'-di(5-acenaphthyl)guanidineon the amplitude of the response to an electrical stimulus.

FIG. 13A depicts a graph showing the results from the bath applicationof adenosine on the field EPSP after electrical stimulation.

FIG. 13B depicts the effect of adenosine on the amplitude of theresponse to an electrical signal.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is related to the discovery that certain substitutedguanidines, amino guanidines and N,N',N",N'"-tetrasubstitutedhydrazinedicarboximidamides have the ability to modulate, i.e. inhibitor potentiate the release of, or lengthen the time course of action of,neurotransmitters from neuronal tissue. As a result, these compounds maybe used to treat or prevent those pathophysiologic conditions whichresult from excessive or inadequate release of neurotransmitters.Although applicants do not wish to be bound by any particular theory, itappears that the substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention mediate the inhibition of neurotransmitter release by blockingpresynaptic calcium channels.

Unexpectedly, the applicants have discovered that there are threedistinct subcomponents of glutamate release from depolarized-stimulatedneuronal cells. As shown in FIG. 1, the first component is a rapidlydecaying Ca²⁺ -dependent phasic component with a decay time constant of<200 msec (termed the phasic component). A second component (termedtonic) is Ca²⁺ -dependent and persists for at least one second andgenerally for the duration of the depolarization. A third component isCa²⁺ -independent and may result from the reversal of electrogenicNa-dependent glutamate uptake system. As shown in FIG. 4, blockage ofthe tonic Ca²⁺ -dependent component correlates well with the blockage ofcalcium uptake. Calcium influx into the nerve terminals is an importantstep in the cascade of changes which occur in neuronal cell death fromischemia. These results suggest that the substituted guanidines, aminoguanidines and N,N',N",N'"-tetrasubstituted hydrazine-dicarboximidamidesof the invention hold promise in the prevention of neuronal death fromischemia.

The inhibition of glutamate release by the compounds of the inventiondoes not appear to be related to any sigma receptor binding activity.Both N,N'-di-(adamantan-1-yl)guanidine andN,N'-di-(adamantan-2-yl)guanidine have similar inhibitory activity onglutamate release (55% and 60% inhibition at 30 μM, respectively) butwidely different sigma receptor binding activities (IC₅₀ =16.5 nM and92.7 nM against ³ H-DTG, respectively).

The identification of three subcomponents of inhibition of glutamaterelease allow for the screening of compounds which exhibit selectivityfor the Ca²⁺ -dependent persistent component of glutamate release. Theidentification of such compounds will provide for more efficacious drugswith fewer side effects.

In general, substituted guanidines which may modulate neurotransmitterrelease and which are useful in the practice in the practice of theinvention include compounds having the Formula (I): ##STR16## wherein Rand R¹ are the same or different and are cycloalkyl of 3 to 12 carbonatoms, e.g., cyclopropyl, cyclopentyl, cyclohexyl, cyclohexenyl,cycloheptyl, 1,4-methylenecyclohexyl, 1- or 2-adamantyl, exo or endo2-norbornyl, exo or endo 2-isobornyl, menthyl, cyclopentyl-methyl,cyclohexylmethyl, 1- or 2-cyclohexylethyl and 1-, 2- or3-cyclohexylpropyl; carbocyclic aryl, alkaryl, aralkyl or heterocyclic,e.g., of 6 to 18 carbon atoms and containing 1-3 separate or fusedrings, and 0-5 O, N and/or S ring atoms in an aryl, alicyclic or mixedring system, e.g., phenyl, benzyl, 1- and 2-phenylethyl, 1-, 2-, or3-phenylpropyl; o-, m-, or p-tolyl, m,m'-dimethylphenyl, o-, m-, orp-ethylphenyl, m,m'-diethylphenyl, m-methyl-m'-ethylphenyl,o-propylphenyl, p-isopropylphenyl, p-tert-butylphenyl, p-n-propylphenyl,p-cyclopropylphenyl, p-cyclohexylphenyl, p-n-butylphenyl; naphthyl, e.g.1-or 2-naphthyl; biphenyl; or a heterocyclic group such as indanyl, e.g.4-indanyl; indenyl, e.g. 1- or 4-indenyl; acenaphthyl, e.g. 3- or5-acenaphthyl; acenaphthylenyl, e.g. 5-acenaphthylenyl; indolyl, forexample, 7-indolyl; benzthiazole, quinolinyl, isoquinolinyl, pyridyl,pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrrolyl, thiazolyl, oxazolyl,imidazolyl and coumarinyl; and

R² and R³ are the same or different and are hydrogen, C₁ -C₆ alkyl,lower C₁ -C₆ alkylamino, aryl or substituted aryl; or R and R₂ or R¹ andR³ form a C₄ to C₆ heterocyclic ring together with the guanidinenitrogen which may be fused to or substituted by a benzene ring.

The groups R, R¹, R² and R³ may be substituted with one or moresubstituents including hydroxy, amino, oxo, lower C₁₋₆ alkyl, C₁ -C₆cycloalkyl, lower C₁ -C₆ alkylamino, C₁ -C₆ alkoxy, nitro, azido, cyano,isocyanato, amido, carbamido, sulfonate, or halogen, e.g. fluorine,chlorine, bromine or iodine.

Particular compounds within the scope of Formula I includeN,N'-di(adamant-1-yl)guanidine, N,N'-di(adamant-2-yl)guanidine,N-(adamantan-1-yl)-N'-(1-naphthyl)guanidine,N-(adamantan-2-yl)-N'-(1-naphthyl)guanidine,N-(adamantan-1-yl)-N'-(2-iodophenyl)guanidine,N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine,N-(adamantan-2-yl)-N'-(2-methylphenyl)guanidine,N-((±)-endo-2-norbornyl)-N'-(2-iodophenyl)guanidine,N-(α-naphthyl)-N'-(2-iodophenyl)guanidine,N-(3-ethylphenyl)-N'-(1-naphthyl)guanidine, N-(1-naphthyl)-N'-(m-ethylphenyl)-N'-methylguanidine,N,N'-di(4-n-butylphenyl)guanidine, N,N'-di(4-cyclohexylphenyl)guanidine,N,N'-di(4-biphenyl)guanidine, N,N'-di(4-neopentylphenyl)guanidine,N,N'-di(4-cyclopropylphenyl)guanidine,N,N'-di(4-isopropylphenyl)guanidine, andN,N'-di(4-tert.-butylphenyl)guanidine.

Preferred disubstituted guanidines within the scope of general Formula(I) include compounds having the Formula (II): ##STR17## wherein theguanidine nitrogen may be substituted at any position of the adamantylgroups;

R² and R³ are as defined above;

X¹ and X² are the same or different and are selected from the groupconsisting of hydrogen, hydroxy, acetate, oxo, amino, lower C₁₋₆ alkyl,amino, alkoxy of 1-6 carbon atoms, e.g., methoxy, ethoxy and propoxy;lower C₁₋₆ alkyl amino, di-lower C₂₋₁₂ alkyl amino, nitro, azido,sulfhydryl, cyano, isocyanato, or halogen, e.g. fluoro, chloro, bromo,or iodo; amido, e.g. , acetamido, N-ethylacetamido; carbamido, e.g.,carbamyl, N-methylcarbamyl, N,N'-dimethylcarbamyl; etc., wherein atleast one of X¹ and X² is other than hydrogen.

Other preferred guanidines within the scope of general Formula (I)include compounds having the Formula (III): ##STR18## wherein R, R¹, R²,and X¹ are as defined above.

Preferred compounds within the scope of general Formula III includeN,N'-di-(1-acenaphthyl)guanidine, N,N'-di-(3-acenaphthyl)guanidine,N,N'-di-(5-acenaphthyl)guanidine, N,N'-di-(acenaphthylen-1-yl)guanidine,N-(adamantan-1-yl)-N'-(adamant-2-yl)-N'-(5-acenaphthyl)guanidine;N-(adamantan-2-yl)-N-(5-acenaphthyl)guanidine;N-(adamantan-1-yl)-N-(3-acenaphthyl)guanidine;N-(adamantan-2-yl)-N'-(3-acenaphthyl)guanidine;N-(adamant-1-yl)guanidine;N-(3-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N-(4-cyanonaphthyl)guanidine;N-(5-acenaphthyl)-N-(4-cyanonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-amidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-amidonaphthyl)guanidine;N-(3-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(5-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(7-fluroronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(7-fluroronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(5-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(3-acenaphthyl)-N'-(4-n-propylphenyl)guanidineN-(5-acenaphthyl)-N'-(4-n-propylphenyl)guanidineN-(3-acenaphthyl)-N-(2-isopropylphenyl)guanidineN-(5-acenaphthyl)-N-(2-isopropylphenyl)guanidineN-(3-acenaphthyl)-N-(4-cyclopropylphenyl)guanidine;N-(5-acenaphthyl)-N-(4-cyclorpopylphenyl)guanidine;N-(3-acenaphthyl)-N'-(coumarinyl)guanidine;N-(5-acenaphthyl)-N'-(coumarinyl)guanidine;N-(3-acenaphthyl)-N'-(quinolinyl)guanidine;N-(5-acenaphthyl)-N'-(quinolinyl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanodine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N,N'-bis(4-bromo-3-acenaphthyl)guanidine;N,N'-bis(4-bromo-5-acenaphthyl)guanidine;N,N'-bis(4-hydroxy-3-acenaphthyl)-guanidine;N,N'-bis(4-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(4-amino-3-acenaphthyl)guanidine;N,N'-bis(4-amino-5-acenaphthyl)guanidine;N,N'-bis(4-nitro-3-acenaphthyl)guanidine;N,N'-bis(4-nitro-5-acenaphthyl)guanidine;N,N'-bis(1-bromo-3-acenaphthyl)-guanidine;N,N'-bis(1-bromo-5-acenaphthyl)guanidine;N,N'-bis(2-bromo-3-acenaphthyl)guanidine;N,N'-bis(2-bromo-5-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(1-oxo-3-acenaphthyl)guanidine;N,N'-bis(1-oxo-5-acenaphthyl)guanidine;N,N'-bis(2-oxo-3-acenaphthyl)guanidine;N,N'-bis(2-oxo-5-acenaphthyl)guanidine;N,N'-bis(3-acenaphthylenyl)guanidine; N,N¹-bis(4-azido-5-acenaphthyl)guanidine; N,N¹-bis(4-sulfonyl-5-acenaphthyl)guanidine; andN,N'-bis(5-acenaphthylenyl)guanidine.

Other substituted guanidines which may modulate neurotransmitter releaseand which are useful in the practice in the practice of the inventioninclude compounds having the Formula (IV): ##STR19## wherein R, R¹, R²and R³ are as described above, X³ and X⁴ have the same meaning as X¹ andX² ; and

x and y are the same or different and are 0, 1, 2, 3 or 4.

Preferred substituted guanidines within the scope of general Formula (I)include compounds having the Formula (V): ##STR20## wherein R², R³, X¹,X², X³, X⁴, x any y are as described above.

Preferred substituted guanidines within the scope of Formula (V) includeN,N'-di-(3-nitroadamantan-1-yl)guanidine,N,N'-di-(3-hydroxyadamantan-1-yl)guanidine,N,N'-di-(3-amino-adamantan-1-yl)guanidine,N,N'-di-(3-nitro-adamantan-2-yl)guanidine,N,N'-di-(3-hydroxyadamantan-2-yl)guanidine,N,N'-di-(3-aminoadamantan-2-yl)guanidine,N,N'-di-(5-nitroadamantan-2-yl)guanidine,N,N'-di-(5-hydroxyadamantan-2-yl)guanidine,N,N'-di-(5-aminoadamantan-2-yl)guanidine,N,N'-di-(methylene-adamantan-1-yl)guanidine,N,N'-di-(methylene-adamantan-2-yl)guanidine,N-(adamantan-1-yl)-N'-(methyleneadamantan-1-yl)-guanidine,N-(adamantan-2-yl)-N'-(methyleneadamantan-2-yl)-guanidine,N-(adamantan-1-yl)-N'-(methyleneadamantan-2-yl)guanidine,N-(adamantan-2-yl)-N'-(methyleneadamantan-1-yl)guanidine,N,N'-di-(methylene-(3-aminoadamantan-1-yl))guanidine,N,N'-di-(methylene-(3-aminoadamantan-2-yl))guanidine,N,N'-di-(methylene-(3-hydroxyadamantan-1-yl))guanidine,N,N'-di-(methylene-(3-hydroxyadamantan-2-yl))guanidine,N,N'-di-(methylene-(3-mercaptoadamantan-1-yl))guanidine,N,N'-di-(methyl-ene-(3-mercapto-adamantan-2-yl))guanidine,N,N'-di-(methyl-ene-(3-mercaptoadamantan-1-yl))guanidine,N,N'-di-(methylene-(3-mercaptoadamantan-2-yl))guanidine,N,N'-di-(methylene-(3-cyanoadamantan-1-yl))guanidine,N,N'-di-(methylene-(3-cyanoadamantan-2-yl))guanidine,N,N'-di-(methylene-(3-cyanoadamantan-1-yl))guanidine andN,N'-di-(methylene-(3-cyanoadamantan-2-yl))guanidine.

Other substituted guanidines which may modulate neurotransmitter releaseand which are useful in the practice in the practice of the inventioninclude compounds having the Formula (VI): ##STR21## wherein R, R¹, R²and R³ are as defined above and R⁴ and R⁵ have the same meaning as R andR¹. Compounds having Formula (V) may be either symmetrical dimers of asingle guanidine or conjugates of different guanidines.

Especially preferred compounds within the scope of Formula (VI) whichmay be used in the practice of the invention includeN,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide,N,N',N",N'"-tetraphenylhydrazinedicarboximidamide,N,N'-di-(adamantan-1-yl)-N",N'"-dicyclohexylhydrazinedicarboximidamide,N,N'-di-(adamantan-2-yl)-N",N'"-dicyclohexylhydrazinedicarboximidamide,N,N',N",N'"-tetra-(adamantan-1-yl)-hydrazinedicarboximidamide,N,N',N",N'"-tetra-(adamantan-2-yl)-hydrazinedicarboximidamide,N,N'-di-(adamantan-1-yl)-N",N'"-di-(adamantan-2-yl)-hydrazinedicarboximidamide,N,N'-di-(2-norbornyl)-N",N'"-dicyclohexylhydrazinedicarboximidamide,N,N'-di-(2-isobornyl)-N",N'"-dicyclohexylhydrazinedicarboximidamide,N,N'-di-(2-isobornyl)-N",N'"-di-(adamantan-1-yl)hydrazinedicarboximidamide,andN,N'-di-(2-isobornyl)-N",N'"-di-(adamantan-2-yl)hydrazinedicarboximidamide.

Other substituted guanidines which may modulate neurotransmitter releaseand which are useful in the practice in the practice of the inventioninclude compounds having the Formula (VII): ##STR22## wherein R, R¹, R²,R³, X³, X⁴, x and y are defined above. Preferred compounds havingFormula (VI) are wherein R and R¹ are adamantyl groups.

Other substituted guanidines which may modulate neurotransmitter releaseand which are useful in the practice in the practice of the inventioninclude compounds having the Formula (VIII): ##STR23## wherein R, R¹ R²and R³ are as described above and R⁶ is hydrogen, C₁ -C₆ alkyl, C₃ -C₁₂cycloalkyl, carbocyclic aryl, nitrile, C₁ -C₆ alkoxycarbonyl, C₁ -C₆acyl or benzoyl.

Other substituted guanidines which may modulate neurotransmitter releaseand which are useful in the practice in the practice of the inventioninclude compounds having the Formula (IX): ##STR24## wherein R, R¹, R²,R³, R⁶, X³, X⁴, x and y are as defined above.

Preferred compounds within the scope of Formula (IX) includeN,N'-dicyclohexyl-N"-aminoguanidine,N,N'-di-(adamantan-1-yl)-N"-aminoguanidine,N,N'-di-(adamantan-2-yl)-N"-aminoguanidine,N,N'-di-(2-norbornyl)-N"-aminoguanidine, andN,N'-di-(2-isobornyl)-N"-aminoguanidine.

Preferably, 0, 1, 2, 3 or more polar groups defined by X¹ and X² may bepresent on the R groups of compounds having Formulae (I)-(IX).Especially preferred are compounds of Formulae (I)-(IX) wherein at leastone of R and R¹ is a 1- or 2-substituted adamantyl group or a 3- or5-acenaphthyl group. Other especially preferred compounds are those withincreased aqueous solubility. Such compounds are where at least one ofX¹ or X² is a polar group or at least one of R and R¹ is substituted bya polar group.

The substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention may exist as any one of a number of tautomeric isomers. Anyone of these tautomeric isomers are within the scope of compounds whichare useful in the claimed invention.

The substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention are effective modulators of neurotransmitter release fromischemic neuronal cells. Modulation of neurotransmitter release involveseither the inhibition of neurotransmitter release, the potentiation ofneurotransmitter release, or the modulation of the time course of actionof neurotransmitters.

The compounds of the invention are effective inhibitors if they cause atleast about a 50% inhibition of neurotransmitter release at aconcentration of about 100 μM according to the protocol disclosedherein. More preferably, the compounds cause at least about a 50%inhibition of neurotransmitter release at a concentration of about 30μM. The compounds of the invention are effective potentiators ofneurotransmitter release if they cause at least about a 50% potentiationof neurotransmitter release at a concentration of about 100 μM. Morepreferably, the compounds cause at least about a 50% potentiation ofneurotransmitter release at a concentration of about 30 μM. Thecompounds of the invention are effective upward or downward modulatorsof neurotransmitter decay kinetics if they cause a doubling or halving,respectively, of the neurotransmitter release kinetics. For example,effective upward modulators of neurotransmitter release kinetics maycause a doubling of decay kinetics from about 100 msec (as observed forthe Ca²⁺ -dependent tonic component of glutamate release) to about 200msec.

The neurotransmitters which may modulate neurotransmitter releaseinclude, but are not limited to glutamate, dopamine, norepinephrine,glycine, aspartate, and serotonin. One of ordinary skill in the art canidentify those compounds which are effective modulators ofneurotransmitter release using the procedures disclosed herein with nomore than routine experimentation.

The most promising compounds for treatment or prevention of neuronaldeath can be evaluated in vivo in one or more variations of the ratmiddle cerebral artery occlusion model. Such models are generallyconsidered to be particularly predictive or neuroprotective efficacy instroke (Ginsburg, M.D. and Busto, R. B., Stroke 20: 1627-1642 (1989)).

In parallel, the efficacy of lead candidates may be assessed in thewidely accepted 4-vessel occlusion model of global ischemia (Pulsinelli,W. A. and Buchan, A. M., Stroke 19:913-941 (1988)). Initially, claspsare placed around each common carotid artery of anaesthetized rats, andthe vertebral arteries are electrocoagulated. After 24 hours, the claspsare tightened for 10-30 minutes and then loosened to allow reperfusion.Three days later, the animals are sacrificed and the brains are examinedhistologically.

Numerous studies have emphasized the importance of administration ofneuroprotective drugs very soon after the ischemic insult (Ginsburg, M.D., and Busto, R. B., Stroke 20:1627-1642 (1989)). Therefore, in bothmodels, candidate compounds may be administered intravenously orintraperitoneally at varying time intervals within 1 hour after theonset of ischemia, to evaluate the optimum window of therapeuticefficacy.

Disubstituted guanidines are the subject of copending application Ser.No. 07/237,367 filed Aug. 29, 1988, and U.S. Pat. No. 4,709,094, whosedisclosures are incorporated herein by reference. The preferredguanidines in U.S. Pat. No. 4,709,094 are described therein by theformula: ##STR25## wherein R and R¹ are each independently alkyl,cycloalkyl, carbocyclic aryl, alkaryl or aralkyl. As a class, thesecompounds are described in this patent as exhibiting a highly selectivebinding activity to the sigma brain receptor. In copending applicationSer. No. 07/237,367, it is disclosed that additional specific members ofthis class of disubstituted guanidines exhibit a high binding activityfor the PCP receptor.

These N,N'-disubstituted guanidines of Formula III can readily beprepared by conventional chemical reactions, e.g., when R and R¹ are thesame, by reaction of the corresponding amine with cyanogen bromide.Other methods which can be employed include the reaction of an aminewith a preformed cycloalkyl or aryl cyanamide. See Safer, S. R., et al.,J. Org. Chem. 13:924 (1948). This is the method of choice for producingasymmetrical N,N'-disubstituted guanidines. For a recent synthesis ofasymmetrical guanidines, see G. J. Durant et al., J. Med. Chem. 28:1414(1985), and C. A. Maryanoff et al., J. Org. Chem. 51:1882 (1986), thedisclosures of which are incorporated by reference herein.

The compounds having Formulae (VI) and (VII) may be prepared by reactinga symmetrical or unsymmetrical carbodiimide with hydrazine in an organicsolvent (see the Examples). The compounds having Formulae (VIII) and(IX) may be prepared by reacting a symmetrical or unsymmetricalcarbodiimide with hydrazine to give a 1:1 adduct. See Example 2; Weygandand Hilgetag in Preparative Organic Chemistry, Hilgetag and Martini(eds.), John Wiley & Sons, New York, N.Y., page 408 (1972); or Vasilev,P. et al., Chem. Abstr. 93: 150095u.

The compounds of the invention bearing R and R¹ groups having polarsubstituents may be prepared using the above-noted methods wherein thestarting material (R--NH₂ or R¹ --NH₂) has a polar group or a protectedform thereof. Methods for preparing such starting materials are taught,for example, in U.S. Pat. No. 4,649,222, Morat and Rassat, Tet.Lett.:4561-4564 (1979); Sollott and Gilbert, J. Org. Chem. 45:5405-5408(1980); and Zajac, W. W. et al., J. Org. Chem. 54:2468-2471 (1989); thedisclosures of which are incorporated by reference herein in theirentirety.

N,N'-disubstituted-N"-aminoguanidines may be prepared according to anyof the methods which are well known in the art. See, for example, GermanPatent No. DE 2,452,691, Sunderdiek, R., et al., Chem. Abstr. 81:91439m(1974), Bent, K. J., et al., Chem. Abstr. 74:63479m (1971), GermanPatent No. 2,029,707, Huisgen et al., Chem. Abstr. 63:2975d (1965),Kroeger, F., et al., Chem. Abstr. 60:9264f, Heinisch, L., J. Pract.Chem. 329:290-300 (1987), Kramer, C.-R., et al., Biochem. Physiol.Pflanzen. 178:469-477 (1983), Prasad, R. N., et al., Can. J. Chem.45:2247-2252 (1967), Huisgen et al., Chem. Ber. 98:1476-1486 (1965),Podrebarac, E. G., et al., J. Med. Chem. 6:283-288 (1963), Kroger etal., Ber. 97:396-404 (1964), and Durant, G. J., et al., J. Med. Chem.9:22-27 (1966).

In a compositional aspect, this invention relates to a pharmaceuticalcomposition in unit dosage form and adapted for systemic administrationto a subject, e.g., a human being, comprising per unit dosage an amountof a substituted guanidine, amino guanidine orN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamide of the inventioneffective to modulate the release of a neurotransmitter. Preferably, theneurotransmitter is glutamate. Such compounds are preferably effectivemodulators of neurotransmitter release from ischemic neuronal cells.

In another compositional aspect, this invention relates to aneuroprotecting substituted guanidine, amino guanidine orN,N',N",N'"-tetrasubstituted hydrazine-dicarboximidamide of theinvention which modulates the release of a neurotransmitter, inparticular glutamate, and the physiologically acceptable salts thereof.Preferably, such compounds are effective inhibitors of neurotransmitterrelease from ischemic neuronal cells.

In a method aspect, this invention relates to a method for treating orpreventing certain neurological disorders, including nausea resultingfrom chemotherapy, the consequences of stroke or traumatic brain injury,epilepsy or neurological diseases, carbon monoxide poisoning, cyanidepoisoning, toxic brain damage caused by, for example, tetrodotoxin orshellfish toxins, anxiety, and river blindness, comprising theadministration of an effective amount of a substituted guanidine, aminoguanidine or N,N',N",N'"-tetrasubstituted hydrazinedicarboximidamide ofthe invention which inhibits the release of a neurotransmitter to asubject in need of such treatment. Such substituted guanidines, aminoguanidines and N,N',N",N'"-tetrasubstituted hydrazinedicarboximidamidesof the invention may be noncompetitive blockers of neurotransmitter(e.g. glutamate) release. Preferably, compounds are effective inhibitorsof neurotransmitter release from ischemic neuronal cells.

In a further method aspect, this invention relates to a method ofameliorating the neurotoxic effect of ischemia, comprising administeringto a subject, e.g., a human being exhibiting symptoms of or susceptibleto such ischemia, a substituted guanidine, amino guanidine orN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamide of theinvention, which inhibits the release of a neurotransmitter, in anamount effective to ameliorate the neurotoxic effect.

In another method aspect, the present invention relates to a method oftreating Korsakoff's disease, a chronic alcoholism-induced condition,comprising administering to a mammal a substituted guanidine, aminoguanidine or N,N',N",N'"-tetrasubstituted hydrazinedicarboximidamide ofthe invention, in an amount effective to treat the disease. Pretreatmentof animals with the substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention may markedly attenuate the extent of cell loss, hemorrhagesand amino acid changes in a rat model of Korsakoff's disease. SeeLanglais, P. J. et al., Soc. Neurosci. Abstr. 14:774 (1988).

In another method aspect, the present invention relates to a method oftreating or preventing HIV-induced dementia and blindness, comprisingadministering to a mammal a substituted guanidine, amino guanidine orN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamide of theinvention, in an amount effective to treat the disease. As disclosed byDreyer et al., Science 248: 364-367 (1990), gp120 neurotoxicity isassociated with increased levels of Ca²⁺ which are apparently mediatedby excitatory amino acids binding at the NMDA receptor site. Therefore,the substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides will findutility in treating or preventing HIV-induced dementia and blindness bypreventing the release of excessive glutamate.

The substituted guanidines, amino guanidines andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides will findutility in treating or preventing conditions treatable by the blockageof sodium ion channels, e.g. epilepsy. Although the mechanism ofblockage of neurotransmitter release by PK 26124 is not yet clearlydefined, this compound may antagonize not the presynaptic calciumchannels but, instead, the sodium channels in or near the nerveterminals. See Hays, S. J. et al., Abstr. 201st Amer. Chem. Soc. Natl.Meeting, Med.Chem. Abstr. No. 14 (1991). In addition, certainantiepileptic agents, for example phenytoin (ibid; McLean and MacDonald,J. Pharmacol. Exp. Ther. 227:779 (1990)) and lamotrigine (Leach, M. J.et al., Epilepsia 27:490-497 (1986)) block sodium channels in ause-dependent fashion. Specifically, they inhibit the ability of neuronsto sustain repetitive firing, and their maximum efficacy occurs undersituations where bursts of sodium channel-mediated action potentialsoccur, such as in epilepsy. N,N'-di(5-acenaphthyl)guanidine is shown inFIG. 7 to block sodium channels, in addition to the presynaptic calciumchannels. This dual action may be a desirable property ofneuroprotective agents, as they may be more effective blockers ofneurotransmitter release. Moreover, the use-dependence, ability to blockrelease of glutamate and other neurotransmitters under conditionscausing sustained depolarization of neurons and/or repetitive firing ofaction potentials, may be desirable properties of neuroprotectiveagents.

The invention also relates to the treatment of amnesia with thecompounds of the invention. The glutamate release blocker riluzole hasbeen shown to prevent memory loss in ischemic animals. Therefore, it isexpected that the disubstituted guanidines,° aminoguanidines, andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention are also useful for treating or preventing memory loss.

The invention also relates to the treatment of migraine with thecompounds of the invention. The calcium channel blocker disclosed in EP0 266 574 reportedly is useful for the treatment of migraine. Therefore,it is expected that the disubstituted guanidines, aminoguanidines, andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention are also useful for treating or preventing migraines.

The invention also relates to the treatment or prevention ofmulti-infarct dementia, a progressive deterioration of brain functionand intellect due to multiple small strokes over time. The disubstitutedguanidines, aminoguanidines, and N,N',N",N'"-tetrasubstitutedhydrazinedicarboximidamides of the invention are expected to be usefulin treating elderly patients who suffer from multi-infarct dementia aswell as arteriosclerosis.

The methods of the present invention may be practiced on any animal,especially mammals and, more particularly, humans. However, it isintended that any animal which may experience benefit fromadministration of the disubstituted guanidines, aminoguanidines, andN,N',N",N'"-tetrasubstituted hydrazinedicarboximidamides of theinvention are within the scope of animals which may be treated accordingto the invention.

The new drug screening method of the invention allows the identificationof drugs which may be antagonists of the presynaptic calcium channel andwhich inhibit certain subcomponents of neurotransmitter release fromneuronal tissue subject to ischemia.

Such compounds which inhibit neurotransmitter release can be determinedby a method involving:

(a) contacting immobilized synaptosomes containing radiolabelledneurotransmitter with a compound suspected of inhibitingneurotransmitter release;

(b) inducing, by depolarization, the release of radiolabelledneurotransmitter from the immobilized radiolabelled synaptosomesobtained in step (a); (c) washing the immobilized radiolabelledsynaptosomes obtained in step (b) with a buffer comprising said compoundand fractionating the effluent every 15 to 500 msec; and

(d) detecting the relative amount of radiolabelled neurotransmitter ineach fraction compared to control synaptosomes which have not beenexposed to the compound of interest;

wherein a reduced amount of released radiolabelled neurotransmitter inthe fractions from synaptosomes treated with the compound relative tocontrol synaptosomes indicates that the compound inhibitsneurotransmitter release.

This drug screening method requires that the entrapped synaptosomes behoused in a superfusion chamber having a very small dead volume to allowsubsecond time resolution. Such superfusion chambers are taught, forexample, in U.S. Pat. No. 4,891,185 and by Turner, T. J. et al., Anal.Biochem. 178:8-16 (1989), the disclosures of which are fullyincorporated by reference herein.

This method also allows for the screening of drugs which selectivelyinhibit a Ca²⁺ -dependent or Ca²⁺ -independent subcomponent ofneurotransmitter release. As shown in FIG. 1, there are both phasic andtonic components of glutamate release in the presence of Ca²⁺ and a Ca²⁺-independent component which can be seen in the absence of Ca²⁺.Preferably, drugs are identified which selectively inhibit the Ca²⁺-dependent tonic component of glutamate release. Such drugs may beidentified by running the assay in the presence of Ca²⁺ and the drug inquestion. Preferably, the concentration of Ca²⁺ is about >50 μM. Morepreferably, the concentration of Ca²⁺ is about 2 mM. The selectiveinhibition of the tonic component of neurotransmitter release indicatesthat the drug may be particularly effective in preventing neuronal lossdue to ischemia and in treating other pathophysiologic conditions whichare the consequence of elevated levels of release of neurotransmitterssuch as glutamate.

Preferably, the synaptosomes are immobilized. Methods for preparingimmobilized cells are taught, for example, in U.S. Pat. Nos. 4,390,627,4,212,943, and 4,337,313, the disclosures of which are fullyincorporated by reference herein. Most preferably, the synaptosomes areentrapped in a filter matrix, as more fully described below, in ahousing with a very small dead volume, and a physiologic buffer ispassed therethrough. The effluent is then fractionated over very shortperiods of time (15 to 500 msec) and the relative amounts ofradiolabelled neurotransmitter (preferably glutamate) in each fractionis determined. A plot of the amount of radiolabelled glutamate over timegives a measure of the ability of the compound to inhibitneurotransmitter release. An example of such a plot is shown in FIG. 2.

A more detailed, but not limiting, protocol is as follows:

(a) contacting brain synaptosomes with radiolabelled glutamate for atime sufficient to allow uptake of the radiolabelled glutamate via theNa-dependent glutamate uptake system, to give radiolabelledsynaptosomes;

(b) preparing a suspension of the radiolabelled synaptosomes obtained instep (a) in a physiologic buffer and passing the suspension through afilter matrix to entrap the radiolabelled synaptosomes;

(c) washing the entrapped radiolabelled synaptosomes obtained in step(b) with a physiologic buffer;

(d) contacting the entrapped radiolabelled synaptosomes obtained in step(c) with a compound suspected of being an inhibitor of glutamaterelease;

(e) depolarizing the membranes of the entrapped radiolabelledsynaptosomes obtained in step (d); (f) washing the entrappedradiolabelled synaptosomes obtained in step (e) with a physiologicbuffer comprising the organic compound of interest and fractionating theeffluent every 15 to 500 msec; and

(g) detecting the relative amount of radiolabelled glutamate in eachfraction compared to control effluent which has not been exposed to thecompound of interest.

Where there is a reduced amount of radiolabelled glutamate in theeffluent fractions of synaptosomes treated with the compound relative tocontrol synaptosomes, the compound is an inhibitor of glutamate release.As discussed above, the assay may be conducted in the presence andabsence of Ca²⁺, and the kinetics of the Ca-dependent component ofneurotransmitter release may be analyzed to determine the effect of thecompound on the three subcomponents of glutamate release.

In the above method, synaptosomes are isolated and radioisotopicallylabeled with radiolabelled neurotransmitter. The radiolabelledsynaptosomes are then entrapped in a filter matrix and allowed toequilibrate with a physiologic buffer solution that flows into thesample chamber through a valve. This valve is then closed simultaneouslywith the opening of a second valve allowing a physiologic solutioncomprising the organic test compound to flow through the filter matrixand contact the entrapped synaptosomes. The second valve is then closedsimultaneously with the opening of a third valve comprising a substancewith or without Ca²⁺, which causes depolarization of the synaptosomemembranes, together with the organic test compound. The third valve isthen closed simultaneously with the opening of the second valve. Duringthe entire superfusion period, the effluent comprising each physiologicbuffer, test compound and radiolabelled glutamate is continuouslyfractionated. This method allows for the rapid kinetic measurement ofradiolabelled glutamate release from synaptosomes. Optionally, one ormore of the test solutions comprising >50 μM Ca²⁺ may be deliveredthrough the third valve.

The synaptosomes may be derived from any animal, including the rat. Theymay be isolated according to Example 3, herein, or according to Gray andWhittaker, J. Anat. 96:79-88 (1962); or Suszkiw et al., J. Neurosci.6:1349-1357 (1986).

Any radiolabelled form of the neurotransmitter may be used in thepractice of the screening assay including ³ H and ¹⁴ C labelledneurotransmitter. Both ³ H and ¹⁴ C labelled glutamate are commerciallyavailable.

The entrapped synaptosomes are contacted with the radiolabelledneurotransmitter for a time sufficient to allow uptake of theradiolabelled neurotransmitter by the synaptosomes. A preferred time isabout 10 minutes.

The physiologic buffer may be any buffer known to those of ordinaryskill in the art which is compatible with synaptosomes. See Example 3(basal buffer); Gray and Whittaker, J. Anat. 96:79-88 (1962); or Suszkiwet al., J. Neurosci. 6:1349-1357 (1986).

Filters which may be used to entrap the synaptosomes are matrices ofrandomly-oriented fibers or beads pressed, wound, or otherwise bondedtogether into a tortuous maze of flow channels (definition obtained fromMillipore Corp. Catalogue and Purchasing Guide, Lit. No. PA085, printedSeptember 1985). A depth filter is capable of trapping the substrate ina three dimensional matrix thereby allowing a maximum amount ofsynaptosomes to be loaded onto the filter without causing a blockage(clogging) of the solution flow. The preferred depth filters are glassfiber filters such as Whatman GF/F filters; ceramic filters can also beused. Glass fiber filters are particularly desirable because of theirflexibility, chemical inertness, and low cost. A Millipore SC filter maybe used to maintain the integrity of glass fiber filters. See Turner, T.J. et al., Anal. Biochem. 178:8-16 (1989), incorporated by referenceherein, for a detailed description of the superfusion system and thefilter configuration for entrapping synaptosomes.

The concentration of test organic compound in the physiologic buffer mayrange from 0 to about 1 mM, more preferably, from 0 to about 100 μM. Ifthe guanidine is not soluble in the physiologic buffer, then anequivalent amount of a soluble pharmacologically acceptable salt of theorganic compound, as disclosed herein, may be utilized. Alternatively,the compounds may be diluted from a suitable organic solvent.

The flow rates of the physiologic solutions and test solutions should beat least 0.1 ml/sec. Preferably, the flow rate is about 1.0 ml/sec.

Induction of the neurotransmitter release may be accomplished bydepolarizing the synaptosome membranes by contact with relatively highconcentrations of cations such as K⁺ or by the application of a voltageacross the synaptosomes. Alternatively, the release of theneurotransmitter may be induced by contacting the synaptosomes withveratramine. The concentration of K⁺ necessary to cause depolarizationmay range from about 10 to about 150 mM. A preferred concentration isabout 110 mM (as KCl). The corresponding transmembrane voltage necessaryto cause depolarization may range from about -40 mV to about 60 mV.

The effluent fractions may be collected using any means which allow thecollection of fractions over periods of time as short as about 15 msec.The fractions may be collected in a series of separate tube or may becontinuously applied to an absorbent substrate such as filter paper.Preferably, the fractions are obtained and collected with thesuperfusion instrument described in the present Examples and disclosedin U.S. Pat. No. 4,891,185, the content of which is fully incorporatedby reference herein. In general, the effluent is collected in vialspositioned as a spiral or a circle on a turntable which rotates at 16,33, 45 and 78 rpm, giving 3.75, 1.82, 1.33 and 0.77 seconds perrotation. Fifty collection vials in each single revolution allows forthe collection of fractions for as long as 66 msec. or as short as 15msec.

The compounds of this invention can be administered intranasally, orallyor by injection, e.g., intramuscular, intraperitoneal or intravenousinjection or by transdermal, intraocular or enteral means. The optimaldose can be determined by conventional means. Because many of thesubstituted guanidines employed in this invention are substantiallywater insoluble, they are ordinarily administered in the protonatedform, e.g., as a pharmaceutically acceptable salt of an organic orinorganic acid, e.g., hydrochloride, sulfate, hemisulfate, phosphate,nitrate, acetate, oxalate, citrate, malate, etc.

The compounds of this invention can be employed in mixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral orintranasal application which do not deleteriously react to the activecompounds. Suitable pharmaceutically acceptable carriers include but arenot limited to water, salt solutions, alcohol, vegetable oils,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds.

For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions as well as suspensions, emulsions,or implants, including suppositories. Ampules are convenient unitdosages.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or a carbohydrate carrier binder or the like,the carrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

Intravenous or parenteral administration, e.g., subcutaneous,intraperitoneal, or intramuscular are preferred. The compounds of thisinvention being particularly valuable in the treatment of mammaliansubjects, e.g., humans. Typically, such subjects include those sufferingfrom or likely to suffer from nervous system dysfunctions resultingfrom, for example, epilepsy or nerve cell degeneration which is theresult of hypoxia, hypoglycemia, brain or spinal chord trauma, or brainor spinal chord ischemia. Typical candidates for treatment include heartattack, stroke, brain or spinal cord injury patients, patientsundergoing major surgery where brain ischemia is a potentialcomplication, patients [divers] suffering from decompression sicknessdue to gas emboli in the blood stream, drowning victims, and patientssuffering from carbon monoxide poisoning, cyanide poisoning, and toxicbrain damage from ingestion of tetrodotoxin or shellfish toxin. Othercandidates include patients suffering from amnesia, migraine, andmulti-infarct dementia. Other candidates for treatment include thosepatients afflicted with neurodegenerative diseases such as Huntington'sdisease, Amyotrophic Lateral Sclerosis, Alzheimer's disease, Down'sSyndrome olivopontocerebellar atrophy, and Korsakoff's disease. Inaddition, HIV-infected individuals may be treated with the substitutedguanidines, amino guanidines and N,N',N",N'"-tetrasubstitutedhydrazinedicarboximidamides of the invention to treat or preventblindness and HIV-associated dementia. Moveover, the substitutedguanidines, amino guanidines and N,N',N",N'"-tetrasubstitutedhydrazinedicarboximidamides of the invention may be administered tothose patients who are susceptible to generalized anxiety disorder (GAD)in order to treat or prevent anxiety.

The compounds of the invention may also be employed in cryopreservationsolutions for tissues, organs and animals. The compounds of theinvention in such solutions may be effective for reducing the amount ofneuronal loss associated with the freezing of tissues, organs andanimals.

It will be appreciated that the actually preferred amounts of activecompounds used will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication and the particular site of administration. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

Like the guanidines of U.S. Pat. Nos. 1,411,713, 1,422,506 and1,597,233, the substituted guanidines and related compounds of thepresent invention may also be used as rubber accelerators.

The invention also relates to radiolabeled derivates of the guanidinesand related compounds of the present invention. Preferably, theradiolabel is ³ H, ¹¹ C, ¹⁴ C, ¹⁸ F, ¹²⁵ I, ¹³¹ I, ¹⁵ N, ³⁵ S or ³² P.These radiolabeled compounds may be used to study the receptorsresponsible for their pharmacologic activity as well as for imaging oftissue samples for the distribution of receptors for these compounds.

The invention also relates to the compounds of the invention in the formof pharmaceutically acceptable salts, e.g. salts with pharmaceuticallyacceptable acids such as hydrochloric, sulfuric, acetic, malic,phosphoric or succinic acid, etc.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

The entire text of all applications, patents and publications citedabove and below are hereby incorporated by reference.

EXAMPLES Example 1 Synthesis of N,N'-(Adamantan-2-yl)guanidineHydrochloride

2-Adamantylcyanamide

2-Adamantanamine hydrochloride (25 g, 133.2 mmol, Aldrich) waspartitioned between 200 mL of toluene and 200 mL of 1N NaOH and themixture stirred for 30 minutes. The layers were separated and theaqueous layer extracted with toluene (75 mL). The combined organiclayers were washed with water (100 mL), dried (MgSO₄) and the resultantfiltrate utilized as is (the amount of 2-adamantanamine was assumed tobe 133 mmol). To this ice cold solution was added dropwise with stirringa solution of cyanogen bromide (8.74 g, 82.5 mmol) in 25 mL of toluene.As the addition proceeded, a precipitate formed. After the addition ofthe cyanogen bromide was completed, the reaction mixture was allowed towarm to room temperature and stir for an additional two hours. Theprecipitate was filtered off and the filtrate concentrated in vacuo toafford 10 g of a light yellow solid. The crude product wasrecrystallized (methanol/water) and the product dried in vacuo (1 torr,over P₂ O₅ /KOH) to give 9.04 g (62% yield) of the desired product.

N,N'-(Adamantan-2-yl)guanidine Hydrochloride

A mixture of 2-adamantylcyanamide (3.4 g, 19.35 mmol) and2-adamantanamide hydrochloride (2.9 g, 15.48 mmol) was heated in an oilbath at 205° C. under an atmosphere of argon. The mixture graduallyfused to a semisolid residue (but never yielded a clear melt) and thensolidified. The reaction mixture was allowed to cool and methanol (30mL) and water (30 mL) were then added to the solid. The mixture wasfiltered and the solid obtained warmed with methanol (150 mL) and theinsoluble material removed by gravity filtration. The filtrate wasclarified with activated charcoal and concentrated in vacuo to afford awhite solid. This material was dissolved in 150 mL of boiling ethanoland the resultant solution concentrated over a hot plate to give 60 mL.On cooling, crystallization occurred to give 3 g of product which wasagain recrystallized, but this time from ethanol and water, to give(after drying in vacuo over P₂ O₅ at 65° C. for 24 hrs) 2.92 g ofN,N'-(Adamantan-2-yl)guanidine Hydrochloride. M.p. >340° C. Anal. calcd.for C₂₁ H₃₄ N₃ Cl: C, 67.08; H, 9.11; N, 11.18. Found C, 67.21; H, 9.26;N, 11.34.

Example 2 Synthesis ofN,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide

DCC (1.03 g, 4.97 mmol) was dissolved in tetrahydrofuran (15 mL).Hydrazine (135 mg, 4.22 mmol) was added via syringe. Stirring 30 hoursat room temperature produced a clear yellow mixture which was evaporatedto a white powder (922 mg). Solution of this powder in diethyl ether,filtration and acidification with HCl (60 mL of saturated Et₂ O) gave ayellow white precipitate (856 mg). Two recrystallizations (EtOH in anEt₂ O chamber) gave white prisms (133 mg, 18%, M.P. 288°-290° C.). Thelow percentage of nitrogen in the elemental analysis of this compoundindicates that two equivalents of DCC have reacted to hydrazine, formingthe title compound.

¹ H NMR ([² H]--CH₃ OH) δ3.21 (s, 4, N-bearing cyclohexyl), 2.0-1.5 (m,44, cyclohexyls). ¹³ C NMR ([² H]--CH₃ OH) δ154.1 (guanidine), 51.7(N-bearing cyclohexyl), 32.4 and 24.8 (cyclohexyls). IR shows peaks at1639 and 1589 cm⁻¹. Elem. Analysis (C₂₆ N₆ H₅₀ Cl₂ :1/2 H₂ O). Theor. C59.30, H 9.76, N 15.96, Found C 58.94, H 9.76, N 16.01.

Example 3 Synthesis of N,N'-Bis(5-acenaphthyl)guanidine hydrobromide

5-Aminoacenaphthene

40 gms (Aldrich lot #CY 02301 HX, containing 15% of the 3-nitro isomer)of 5-nitroacenaphthene was dissolved in a mixture of tetrahydrofuran(150 ml) and acetic acid (25 ml.). To this solution was added 1.0 of 10%Pd/C and the mixture hydrogenated (40 psi) at room temperature. After 2hours, the mixture was filtered through a bed of celite and the filtrateconcentrated in vacuo to give a mixture of a solid which darkened (topurple) considerably on exposure to air. This material was redissolvedin methylene chloride and decolorized with activated charcoal and thenthe product recrystallized from a mixture of cyclohexane:ethyl acetate(3:1) to give 8.3 g of clean material--only the major 5-amino isomerbeing present by TLC. This material was used as is directly in the nextstep.

N,N'-Bis(5-acenaphthyl)guanidine hydrobromide

To a stirred solution of 5-aminoacenaphthene (8.1 g, 49 mmol) in ethanol(35 ml) was added, dropwise with stirring, a solution of cyanogenbromide (2.6 g, 24.5 mmol) in ethanol (25 ml). The mixture was thenrefluxed for 6 h and allowed to stand for 48 h for convenience. Thecrystals were collected by filtration and washed with a little ethanoland then ether to give 7.7 g of crude product. This material wascombined with a smaller 1.1 g lot and the total amount dissolved inboiling ethanol (approximately 1 L) and decolorized with activatedcharcoal. The charcoal was removed by gravity filtration and thefiltrate concentrated over a hot plate to 200 mL. On cooling, off whitecrystals were deposited which were dried in vacuo at 100° C. under 1torr, to give 5.9 gms;

mp 288°-290° C.

Anal. Calcd. for C₂₅ H₂₂ N₃ Br(444.35); C, 67.56; H, 4.99; N, 9.46.

Found: C, 67.46; H, 5.06; N, 9.21. HPLC (reverse phase): MeCN/H₂ O(50:50 with 0.1% TFA), single component Rt=12.91 minutes

Example 4 Synthesis of N,N'-Bis(3-acenaphthyl)guanidine hydrobromide

3-Aminoacenaphthene

A 25 g sample of commercial 5-nitroacenaphthene (containing 15% of the3-nitro isomer) was chromatographed (600 g silica gel, 230-400 mesh,hexane/ethyl acetate (10:1)) to give 400 mg of the 3-nitro isomer. Theremainder of the fractions were a mixture of both isomers and thismethod was abandoned as a method to separate the two isomers. The 400 mgsample was hydrogenated (40 PSI, 10% Pd/C, 5% acetic acid in THF) on aParr apparatus to give the amino compound which solidified after removalof the solvent in vacuo. This material was used directly, in the nextstep.

N,N'-Bis(3-acenaphthyl)guanidine Hydrobromide

To a solution of 3-aminoacenaphthene in 15 mL of absolute ethanol wasadded 85 mg of cyanogen bromide. The solution was heated at reflux,under an nitrogen atmosphere, for 11 hours. The solution wasconcentrated in vacuo to ˜8 mL and the mixture cooled in an ice bath togive 120 mg of crude product. This material was recrystallized fromethanol to give 60 mg of product, mp 291°-293° C. The HPLC of thismaterial showed the presence of a single impurity (15%). Therefore, thismaterial was partitioned between a 10% aqueous solution of sodiumbicarbonate and ethyl acetate, with the intention of converting it tothe free base and then running a prep TLC. However, a good portion ofthe material formed an emulsion in the organic layer. This solid wascollected by filtration and turned out to be free of the impurity. Itwas redissolved in 0.7M HCl in methanol and concentrated in vacuo toafford a cream white solid; wt 20 mg; mp 210°(s)-230° C. (slow dec).This step should probably have been omitted, since it is hard to predictif the material is a hydrochloride or hydrobromide. Since we have such asmall quantity of material, and it is clean by TLC and HPLC we made noattempt to carry out an elemental analysis.

HPLC: C18 Reverse phase, MeCN:H₂ O 50:50 with 0.1% TFA, Rt=16.7 min(98.85%), impurities at 4.62 min (0.625%) and 15.83 min (0.517%).

Example 5 Synthesis of N-(3- and5-Acenaphthyl)-N'-(4-isopropylphenyl)-guanidine.HCl

A mixture of 3- and 5-nitroacenaphthene (5 g) was dissolved in EtOAc andplaced into a hydrogenating flask containing Palladium on activatedcarbon (10%, 0.5 g). The reaction mixture was hydrogenated at 40 psi for1.5 hr. The pressure of hydrogen dropped due to the consuming of thehydrogen, and it was brought back to 50 psi a few times during thereaction. When all the starting material was used, the hydrogen wasreleased and the resulting solution was filtered through a pad of celiteand washed with EtOAc (20 mL). The clear filtrate was concentrated anddried under vacuum to yield a mixture of 3- and 5-aminoacenaphthene as abrown solid (4.2 g, 100% yield).

TLC (SiO₂, CH₂ Cl₂): R_(f) 0.2

3- and 5-aminoacenaphthene.HCl

Mixture of 3- and 5-aminoacenaphthene (0.8 g) in MeOH (40 mL) wastreated with HCl/MeOH (0.5M, 20 mL), and the reaction mixture wasstirred at room temperature for 30 min. Then the solution wasconcentrated and dried under vacuum to yield a mixture of 3- and5-aminoacenaphthene.HCl as a gray solid.

Isopropylphenyl cyanamide

A solution of cyanogen bromide (1.59 g, 15 mmol) in diethylether (20)was added dropwise to a solution of 4-isopropylaniline (3.24 g) indiethylether (50 mL) at 4° C. with stirring. After the addition, thereaction mixture was kept stirring at room temperature for 18 h. Then asolution with white precipitates formed and the precipitates wereremoved by filtration. The etherate solution was washed with aqueous HCl(1N, 30 mL, two times) as well as brine, dried over MgSO₄, filtered,concentrated, and dried under vacuum to yield 4-isopropylphenylcyanamide (2.0 g, 84%).

TLC (SiO₂, CH₂ CL₂): R_(f) 0.2 IR(CH₂ CL₂): 2220 cm⁻¹.

Synthesis of Di-(3- and5-Acenaphthyl)-N'-(4-isopropylphenyl)-guanidine.HCl

A stirred solution of 4-isopropylphenyl cyanamide (160 mg) inchlorobenzene (35 mL) as added a mixture of 3- and5-aminoacenaphthene.HCl (230 mg, 1 mmol) at 25° C. under Argon. Thereaction mixture was heated at 150° C. in an oil bath for 20 hr. Ahomogenous solution was obtained and this solution was concentrated todryness by rotavap. The obtained product was further purified by prep.TLC to yield N-(3- and5-Acenaphthyl)-N'-(4-isopropylphenyl)-guanidine.HCl as a light brownsolid (0.28 g, 71% yield). TLC (SiO₂, CH₂ CL₂ /MeOH=9/1): R_(f) =0.36IR(CH₂ CL₂): 1660 cm⁻¹ (guanidine peak).

¹ H NMR(CD₃ OD) ppm: 1.07 (d, J=8.57 Hz, 6H), 2.76 (m, 1H), 3.24 (m,4H), 7.08-7.52 (m, 9H).

¹³ C NMR(CD₃ OD) ppm: 24.30, 30.94, 31.54, 35.01, 118.48, 120.35,121.35, 126.45, 126.86, 127.61, 128.38, 128.98, 129.53, 130.24, 133.85,141.63, 147.99, 148.76, 149.68, 149.87, 157.30.

High Resolution Mass Spec: C₂₂ H₂₃ N₃ 329.1892 (Calc.), 329.1889 (Exp.).

Example 6 Synthesis of N-(3- and5-Acenaphthyl)-N-(4-fluoronaphthyl)-guanidine.HCl

3- and 5-Acenaphthyl cyanamide

A solution of cyanogen bromide in CH₃ CN (5M, 5.1 mL, 25.6 mmol) wasadded slowly into a stirred solution of 3- and 5-aminoacenaphthene (41mmol) in diethylether (65 mL) and EtOAc (10 mL) at 0° C. After theaddition, the reaction mixture was stirred at room temperature for 23 h.Finally, the reaction mixture became a green solution with grayprecipitates; the precipitates were removed by filtration. The greenfiltrate was further washed by aqueous HCl (1N, three times), water (100mL, and brine (200 mL). Then the solution was dried over MgSO₄,filtered, concentrated, and dried under vacuum to yield 3- and5-acenaphthyl cyanamide. The product was a brown solid (3.64 g; 92% inyield).

TLC (SiO₂, CH₂ Cl₂ /MeOH=9/1): R_(f) =0.67.

4-Fluoro-1-aminoaphthalene

4-Fluoro-1-nitro-naphthalene (1 g) was dissolved in EtOAc (20 mL) andplaced into a hydrogenating flask containing Palladium on activatedcharcoal (10%, 0.3 g). The reaction mixture was hydrogenated at 50 psifor 5 hr. During the reaction, the pressure of the hydrogen dropped dueto the consuming of the hydrogen, and it was brought back to 50 psi afew times. Finally, the reaction stopped when all the starting materialwas used. The resulting solution was filtered through a pad of celiteand washed with EtOAc (20 mL). The clear filtrate was concentrated anddried under vacuum to yield 4-Fluoro-1-aminonaphthalene.

TLC (SiO₂, CH₂ Cl₂): R_(f) =0.33

4-Fluoro-1-aminoaphthalene.HCl

To 4-fluoro-1-aminoaphthalene in MeOH (5 mL) was added HCl MeOH (0.5M,20 mL), then the reaction mixture was stirred at room temperature for 30min. A white precipitate formed during the reaction; the precipitate wascollected by filtration and dried under vacuum to yield4-fluoro-1-aminoaphthalene.HCl as a white solid (0.81 g; 80% yield).

N-(3- and 5-Acenaphthyl)-N'-(4-fluoronaphthyl)guanidine.HCl

A mixture of 3- and 5-acenaphthyl cyanamide (516 mg, 2.66 mmol) and4-fluoro-1-aminonaphthalene.HCl (500 mg, 2.53 mmol) in chlorobenzene (15mL was heated to 150° C. for 24 hr with stirring. After the reactionmixture was cooled to room temperature, diethylether (50 mL) was addedand a white precipitate formed. The precipitate was collected byfiltration, redissolved in methanol, treated with Norit for 30 min,filtered and concentrated to yield the crude product. The crude productwas further recrystallized in EtOH/Et₂ O to yield pure (N-(3- and5-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine.HCl (0.52 g, 53% yield).

TLC (SiO₂, CH₂ Cl₂ /MeOH=9/1): R_(f) =0.37

¹ H NMR (CD₃ OD) ppm: 3.33-3.38 (m, 4H), 7.22-8.10 (m, 11H).

¹³ C NMR (CD₃ OD) ppm: 30.97, 31.55, 110.64, 118.41, 120.39, 121.45,122.09, 123.38, 125.84, 127.23, 127.68, 128.02, 128.44, 128.97, 129.56,129.84, 130.37, 133.18, 141.67, 148.09, 149.19, 158.43, 160.13 (d,j=253.4 Hz).

High Resolution Mass Spec: C₂₃ H₁₈ N₃ F 355.1484 (Cacl.), 355.1479(Exp.).

Example 7 Synthesis of N-(3- and4-Acenaphthyl)-N-(4-methoxynaphthyl)guanidine.HCl

4-Methoxy-1-aminonaphthalene

A mixture of 4-methoxy-1-nitro-naphthalene (2 g, 9.84 mmol) wasdissolved in EtOAc (100 mL) and placed into a hydrogenating flaskcontaining Palladium on activated carbon (10%, 0.2 g). The reactionmixture was hydrogenated at 50 psi for 1.5 hr. During the reaction, thepressure of the hydrogen dropped a few times due to the consuming of thehydrogen, and the pressure was brought back to 50 psi. When the startingmaterial was used, the reaction was stopped and the hydrogen wasreleased. The resulting solution was filtered through a pad of celiteand washed with EtOAc (20 mL). The clear filtrate was concentrated anddried under vacuum to yield 4-Fluoro-1-aminonophthalene (100% yield).

TLC (SiO₂, CH₂ Cl₂): R_(f=) 0.24

4-Methoxy-1-aminonaphthalene.HCl

4-Methoxy-1-aminonaphthalene (0.53 g, 3.1 mmol) was dissolved inHCl/MeOH (0.5M, 15 mL), then the reaction mixture was stirred at roomtemperature for 60 min. The reaction mixture was concentrated to drynessto yield the crude product as a solid. The solid was further washed byEtOAc, collected by filtration, and dried under vacuum to yield4-methoxy-1-aminonaphthalene.HCl as a white solid (0.61 g; yield: 96%).

N-(3- and 5-Acenaphthyl)-N'-(4-methoxynaphthyl)guanidine.HCl

Mixture of 3- and 5-acenaphthyl cyanamide (243 mg, 1.25 mmol and4-methoxy-1-aminonaphthalene.HCl (250 mg, 1.19 mmol) in chlorobenzenewas heated to 150° C. for 7 hr with stirring. After the reaction mixturewas cooled to room temperature, it became a mixture of a brown solutionand white precipitates. The precipitates were collected by filtration,washed thoroughly with CH₂ Cl₂ (30 mL) as well as EtOAc (15 mL), anddried under vacuum to yield N-(3- and5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine.HCl(0.41 g, 86% yield) asa white powder.

TLC (SiO₂, CH₂ Cl₂ /MeOH=9/1): R_(f) =0.33

¹ H NMR (CD₃ OD) ppm: 3.32-3.36 (m, 4H), 3.96 (s, 3H), 6.89-8.24 (m,11H).

¹³ C NMR (CD₃ OD) ppm: 30.96, 31.55, 104.76, 118.44, 120.37, 121.42,122.79, 123.64, 123.82, 123.86, 127.08, 127.31, 127.77, 128.32, 128.87,128.98, 130.06, 132.62, 141.67, 148.07, 149.13, 157.62, 158.57.

High Resolution Mass: C₂₄ H₂₁ N₃ O 367.1685 (Cacl.). 367.1686 (Exp.).

Example 8 Basic Protocol for the Preparation of Synaptosomes andSuperfusion

Solutions:

0.32M sucrose solution

0.80M sucrose solution

Basal buffer is of the following composition:

NaCl 147 mM

KCl 3 mM

HEPES 10 mM

Dextrose 10 mM

MgCl₂ 1.2 mM

EGTA-TRIS 1 mM

pH 7.4

High-K⁺ buffer

NaCl 95 mM

KCl 55 mM

The rest of the components in high-K⁺ buffer are of the same compositionas Basal buffer. For studying total ³ H-glutamate release, Ca⁺² is addedto the high-K⁺ buffer at 2.4 mM.

To study the release of presynaptic ³ H-glutamate, synaptosomes fromyoung rats were used. CD Male rats of 4 to 6 weeks (50-75 g) were usedfor the preparation of synaptosomes. Rats were killed by decapitationwith guillotine and the skull bone was opened in the center with thepointed blade of dissection scissors. The bone is then peeled away witha bone cutter and the brain is pried with a micro spatula. Thecerebellum is removed and the rest of the brain is placed in 35 ml of0.32M Sucrose solution and homogenized in a Thomas glass teflonhomogenizer C at the maximum power setting (about 450 rpm) with 16strokes. The pestle is rinsed with 5 ml of sucrose solution and added tothe homogenate. The homogenate is centrifuged for 10 min at 3500 rpm(1500 g) in a SS-34 rotor in Sorvall RC-5B. The resulting pellet (P₁) isdiscarded and the supernatant (S₁) is recentrifuged for 20 min at 8700rpm (8500 g). The supernatant (S₂) is discarded and the pellet (P₂) isresuspended in 5 ml of 0.32M sucrose and hand homogenized with 4 strokes(Thomas C) and the volume was made up to 8 ml. This homogenate waslayered on 20 ml of 0.8M sucrose solution in two centrifuge tubes andspun for 25 minutes at 8700 rpm (8500 g). At the end of this spin, mostof the myelin stays at the interphase of 0.32M and 0.8M sucrose andmitochondria pellet as a brown pellet. The synaptosomes are dispersed inthe 0.8M sucrose. Using a 10 ml pipette, the 0.8M sucrose layer iscollected (without disturbing the top myelin layer or the pellet) anddiluted slowly with an equal volume of chilled basal buffer, whilestirring gently with a Pasteur pipette. This diluted solution iscentrifuged for 10 min at 10,000 rpm (12,000 g) and the pellet isresuspended in 1.5 ml of basal buffer and hand-homogenized in a Wheatonglass-glass 7 ml homogenizer with 8 strokes.

DILUTION OF SYNAPTOSOME PREP FOR SUPERFUSION

The synaptosomal homogenate is diluted 1 ml to 10 ml with basal buffer.This dilution gives a protein concentration of 500 to 600 μg/ml. Onehundred μl of this diluted prep with 50-60 μg of protein is used foreach superfusion event. This amount of protein is found to be optimumfor a rapid washout of released tracer. The diluted synaptosomalsolution was kept on ice until the end of the experiment.

LOADING SYNAPTOSOMES WITH ³ H-GLUTAMATE

³ H-glutamate (purchased from NEN) is stored in the refrigerator.Pipette 6 μl (6 Ci; 40 to 55 Ci/mmol) into a disposable test tube. Add100 μl of diluted prep and mix gently on vortex and incubate at 30° C.for 10 min. ³ H-glutamate is taken up by sodium-coupled glutamatetransporter present in synaptosomal membranes. After 10 min, dilute theincubation mixture with 690 μl of basal buffer. This solution is thentransferred in Tygon tubing that is connected to the synaptosome loadingchamber. Using a syringe filler with air, the solution is forced throughthe Millipore SC--GF/F--Millipore SC filter sandwich. These filtersretain the synaptosomes and the solution flows through.

LOADING CHAMBER

The loading chamber is assembled from a "General Valve" tube-tubeconnector. One end is connected to a barbed fitting which in turn isconnected to a 12 gm Tygon tube and serves as inlet to the chamber. Onthe other end of the connector is attached to the filter assembly. Thefilter assembly consists of a stainless steel washer with a flat sidefacing the filter, a SC Millipore filter, GF/F Whatman glass fiverfilter disc with grid facing into the flow, another SC filter followedby a stainless steel screen and a stainless steel washer with the flatside facing the screen. This whole assembly is secured with a teflonnut. After the synaptosomes are loaded, by unscrewing the nut andtapping it to the workbench, the filter sandwich can be dislodged.

SUPERFUSION

An important component of the superfusion setup is the custom madestainless steel "General Valve" three-valve solenoid operatedsuperfusion chamber with an outlet for the effluent. The three inletsare connected to stainless steel buffer reservoirs that contain (1) washbuffer, (2) control buffer and (3) experimental buffer which are keptunder 40 psi of nitrogen gas through teflon tubing connections. Thesuperfusion is controlled through a menu-driven program on an Apple IIecomputer. See Turner, J. T. et al., Anal. Biochem. 178:8-16 (1989), andU.S. Pat. No. 4,891,185, the disclosures of which are fully incorporatedby reference herein.

The superfusion chamber has a teflon bushing and a stainless steelwasher fixed with the flat surface facing the filters. A GF/B filterdisc is placed first in the chamber to eliminate the switching relatedartifacts. The synaptosome loaded sandwich is then placed facing thesynaptosomes into the flow followed by RA filter, stainless steel screenand a washer with the flat side facing the screen. This assembly issecured with a teflon nut that has two layers of teflon tubing fixedinside with epoxy to reduce the dead volume and improve the effluentflow.

The time protocol can be varied as for the requirements of theparticular experiment. Before superfusion the synaptosomes are subjectedto two 5 sec washes with basal buffer, with a flow rate of about 1.2ml/sec and the effluent is collected for radioactive disposal. After thewash, the outlet is centering on the fraction collection vials. Atypical protocol (with 16 rpm) consists of a first 1.08 sec superfusewith basal buffer and switch to a high-K⁺ buffer for 2.1 sec and aswitch back to basal buffer for the remaining time. By selecting "Do thesuperfusion" from the menu and activating by returning, the effluentflows into vials on a rotating turntable platter.

EFFLUENT COLLECTION

This is done using a turntable which rotates at 16, 33, 45 and 78 rpm,giving 3.75, 1.82, 1.33 and 0.77 seconds for each rotation. On theturntable a platter with 50 uniformly drilled holes in a circle to allowfor glass vials for collecting the effluent. So, depending on the rpm ofturntable, one can collect fraction of as long as 66 msec or as short as15 msec.

The superfusion event and the fraction collection are synchronized witha magnetic reed switch that is activated by a permanently fixed magnetto the turntable. If the flow rate is 1.25 ml/sec, at 33 rpm eachfraction is 45 ul.

To the fractions, one ml of "Hydrofluor" (National Diagnostics,Manville, N.J.) is added, and the fractions are counted in a liquidscintillation counter. After superfusion, all the filters in the chamberare taken out and suspended in 1 ml of counting fluid and counted afterplacing it overnight on a shaking platform and counted the next day.

DATA ANALYSIS

Lotus 123 spreadsheet software is used to analyze the data. The liquidscintillation counter gives the averaged cpm directly. Using aspreadsheet, the counts are entered against time (and fraction number).In the next column, the counter background is subtracted for eachfraction. All of the counts are summed, including the ones retained onthe filter. This represents the total amount taken up by thesynaptosomes. From this sum, each fraction is calculated as a percentrelease and plotted against time. This plot would clearly show thepattern of baseline, valve opening and tracer release. Plotting thesenet results against valve 3 time gives the net release of radiolabelledguanidine in response to experimental buffer as a function of time.

FIG. 1 depicts a graph showing ³ H glutamate release from rat brainsynaptosome which had been loaded with ³ H-glutamate for 10 minutes at30° C. The synaptosomes (50 μg protein) were loaded into the superfusionchamber and washed for 10 seconds with the basal buffer containing 145mM NaCl and 5 mM KCl. The superfusion was then started immediately. Attime 0, the synaptosomes were depolarized by switching to a high Kbuffer (40 mM NaCl, 110 mM KCl). The upper curve in FIG. 1 denotesrelease evoked in the presence of 2.4 mM Ca²⁺. The lower curve displaysrelease evoked in Ca-free buffer. The data are expressed as a percent ofthe total ³ H-glutamate pool released in each 72 millisecond fractioncollected. Error bars are for triplicate determinations.

Example 9 Assay for Inhibition of Glutamate Release

The experimental drugs are first dissolved in methanol to make a stockof 20 mM. This solution is diluted into the basal buffer as well ashigh-K⁺ buffer to give the required concentration of this drug. Allsolutions including the controls are made to have the same concentrationof methanol. Methanol concentration never exceeded 0.3% (v/v) ofbuffers. Synaptosomes were first exposed to the drugs during the washbefore superfusion and also during the entire superfusion protocol. Thetotal time synaptosomes were exposed to the test organic compoundsbefore the glutamate release was <25 sec.

FIG. 2 depicts a graph showing the effect of 0 uM, 10 uM, 30 uM and 100uM N,N'-di-(adamantan-1-yl)guanidine on the release of ³ H-glutamatefrom rat brain synaptosomes. The same concentrations of the guanidinewere maintained in all superfusion solutions. The results are plotted interms of cumulative amount of glutamate release in a single 2.1 secondK⁺ depolarizing pulse, as the percent of total radioactive glutamateloaded in the synaptosomes. As is clear from FIG. 2, increasingconcentrations of N,N'-di(adamantan-1-yl)guanidine resulted indecreasing release of glutamate from synaptosomes.

Following the above procedure, a number of additional compounds werescreened for glutamate-release inhibitory activity. The relative levelsof glutamate release in the presence of these compounds of the inventionappear in Table I.

                  TABLE I                                                         ______________________________________                                        Compound              30 μM.sup.1                                                                        100 μM.sup.1                                 ______________________________________                                        N,N'-di-(o-tolyl)guanidine                                                                          .sup. nd.sup.2                                                                        91                                              N,N'-di-(2-iodophenyl)guanidine                                                                     nd      61                                              N,N-di-(3-methylphenyl)guanidine                                                                    nd      106                                             N-cyclohexyl-N'-(2-methylphenyl)-                                                                   100     nd                                              guanidine                                                                     N-(adamantan-1-yl)-N'-cyclohexyl-                                                                   120     nd                                              guanidine                                                                     N,N-di-(adamantan-1-yl)guanidine                                                                    45      13                                              N-(adamantan-1-yl)-N'-(2-iodo-                                                                      39      nd                                              phenyl)guanidine                                                              N-(adamantan-1-yl)-N'-(2-methyl-                                                                    52      45                                              phenyl)guanidine                                                              N-(adamantan-2-yl)-N'-(2-iodo-                                                                      74      nd                                              phenyl)guanidine                                                              N-(adamantan-2-yl)-N'-(2-methyl-                                                                    100     nd                                              phenyl)guanidine                                                              N-((±)-endo-2-norbornyl)-N'(2-                                                                   77      nd                                              iodophenyl)guanidine                                                          N,N'-di-(adamantan-2-yl)guanidine                                                                   40      20                                              N,N'-di-(1-naphthyl)guanidine.                                                                      19      29                                              N-(α-naphthyl)-N'-(2-iodophenyl)-                                                             77      nd                                              guanidine                                                                     N-(3-ethylphenyl)-N'-(1-naphthyl)-                                                                  59      nd                                              guanidine                                                                     Di-(o-methyl)benzyl)amine                                                                           100     nd                                              N,N'-di-(3-ethylphenyl)-N-methyl-                                                                   88      nd                                              guanidine                                                                     N,N',N",N"'-tetracyclohexylhydra-                                                                   51      nd                                              zinedicarboximidamide                                                         N-(1-naphthyl)-N'-(o-isopropyl-                                                                     100     nd                                              phenyl)guanidine                                                              N-(1-naphthyl)-N-Methyl-N'-(o-iso-                                                                  95      nd                                              propylphenyl)guanidine                                                        N-(1-naphthyl)-N'-(m-ethylphenyl)-                                                                  76      21                                              N'-methylguanidine                                                            Control               100     100                                             ______________________________________                                         .sup.1 Concentration.                                                         .sup.2 nd = not done.                                                    

FIG. 3 depicts a graph showing the effect of 0 uM, 1 uM, 3 uM and 10 uMof N,N'-di(5-acenaphthyl)guanidine over time on the Ca²⁺ -dependentrelease of [³ H] glutamate from radiolabelled synaptosomes after K⁺depolarization (at time 0). FIG. 3 indicates that this compound is apotent inhibitor of glutamate release from brain nerve terminalpreparations. At 3-10 uM concentrations, it blocks the persistentcomponent of Ca²⁺ -dependent glutamate release more than the initialtransient component.

Example 10 Correlation Between Inhibition of Ca Uptake with Inhibitionof Glutamate Release

Several compounds were also tested to determine whether the Ca²⁺dependent and independent components of glutamate release are related tothe blockage of ⁴⁵ Ca uptake. Calcium uptake is one step in the cascadeof events which occur in neuronal cell death from ischemia. SeeBassaclough and Leach, Current Patents Ltd., 2-27 (19₋₋). The Ca-fluxprotocol is as follows. Rat brain synaptosomes were prepared accordingto Hajos, Brain Res. 93:485 (1975). Synaptosomes were suspended in lowpotassium "LK" buffer (containing 3 mM KCl) at 2 mg/ml. Drugs in LK wereadded to synaptosomes to a final concentration of 10 μM and incubatedfor 5 min at room temperature. ⁴⁵ Ca uptake was then measured by addingisotope in either LK or high potassium (150 mM KCl) containing buffer.After 5 seconds, the ⁴⁵ Ca flux was stopped with 0.9 ml quench solution(LK+10 mM EGTA). The solution was filtered under vacuum and the filterswashed with 15 ml of quench buffer. The effect of drug is expressed as %inhibition (or block) of control potassium-stimulated ⁴⁵ Ca influx. Thismethod is an adaptation of the method disclosed by Nachsen andBlaustein, J. Physiol. 361:251-268 (1985).

As shown in FIG. 4, there is a near linear correlation between theinhibition of synaptosomal ⁴⁵ Ca uptake and inhibition of the persistentcomponent (Λ)of glutamate release byN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine (#1),N-(1-naphthyl)-N'-(m-ethylphenyl)-N'-methylguanidine (#2),N,N'-di-(1-naphthyl)guanidine (#3), N,N'-diadamantan-1-yl)guanidine(#4), N,N'-di-(adamantan-2-yl)guanidine (#5),N,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide (#6) andN,N'di-(5-acenaphthyl)guanidine. In contrast, there was no correlationbetween the inhibition of synaptosomal ⁴⁵ Ca uptake and the inhibitionof the phasic component (o) of glutamate release (FIG. 4). An exceptionis N,N'-di(5-acenaphthyl)guanidine which inhibits the persistentcomponent of glutamate release to a greater degree than predicted by itsability to inhibit ⁴⁵ Ca uptake.

FIG. 6 depicts a graph showing the inhibition of potassium-stimulated Cauptake of synaptosomes by N,N'-di-(adamantan-2-yl)guanidine (▪),N,N'-di-(1-naphthyl)guanidine (♦; .sup....... IC₅₀ =9.1 μM, ⁻⁻⁻⁻⁻ IC₅₀=16 μM), N,N',N",N'"-tetracyclohexylhydrazinedicarboximidamide (;₋₋.sup..₋ IC₅₀ =3.3 μM), and N,N'-di-(adamantan-1-yl)guanidine (Δ; ₁₃IC₅₀ =6.6 μM) against the percent of Ca uptake compared to controlsynaptosomes. As shown in FIG. 6, the drugs caused an inhibition ofpotassium-stimulated Ca uptake in a dose-dependent manner.

A number of additional compounds (at 10 μM) were tested for activity ininhibiting/potentiating the potassium-stimulated uptake of calcium intosynaptosomes. The results are shown in Table II:

                  TABLE II                                                        ______________________________________                                        Compound              % Uptake vs. Control                                    ______________________________________                                        N-(α-Naphthyl)-N'-(1-                                                                         131                                                     piperidinyl)-guanidine                                                        N-(Cyclohexyl)-N'-(1- 108                                                     piperidinyl)-guanidine                                                        N-(α-Naphthyl)-N'-(4-                                                                         79                                                      phenylpiperidinyl)-guanidine                                                  N,N'-Bis(indan-1-yl)- 88                                                      guanidine                                                                     N,N'-Bis(m-ethylphenyl)-2-                                                                          101                                                     imino-imidazolidine                                                           N,N'-Bis(o-tolyl)-N,N'-                                                                             119                                                     butanyl(bridge)-guanidine                                                     N-N'-Bis(1-Adamantanemethyl)-                                                                       111                                                     guanidine                                                                     N-(8-Aminocoumarinyl)-N'-(m-                                                                        151                                                     ethylphenyl)-N'-methyl-                                                       guanidine                                                                     N,N'-Bis(1,2,3,4-tetrahydro-                                                                        87                                                      1-quinolinyl)-guanidine                                                       N,N'-Bis(5-acenaphthyl)-                                                                            43                                                      guanidine                                                                     N-(m-Ethylphenyl)-N'-(2-                                                                            71                                                      carboethoxy-7-benzofuranyl)-                                                  N-methyl guanidine                                                            N-(α-Naphthyl)-N'-(m-                                                                         95                                                      ethylphenyl)-N"-cyanoguanidine                                                N-(Adamantan-1-yl)-N'-(4,5-benzo-                                                                   98                                                      2-thia-1,3-diazol-6-yl)guanidine                                              ______________________________________                                    

Table III shows the percentage inhibition of synaptosomal ⁴⁵ Ca uptakeby certain N,N'-disubstituted guanidines of the invention.

                  TABLE III                                                       ______________________________________                                        Inhibition of Synaptosomal .sup.45 Ca Uptake                                  By Substituted Guanidines and Related Compounds                                                 Inhibition of .sup.45 Ca                                                      Uptake                                                                          %                                                                             Inhibition                                                                    @ 10 uM   IC50,                                           Compound            (SEM)     uM                                              ______________________________________                                        N,N'-Bis(5-acenaphthyl)guanidine                                                                  46 (±15)                                                                             10.7   ± 3                                   N-(Adamantan-1-yl)-N'(α-naphthyl)                                                           31 (±14)                                                                             --                                              guanidine                                                                     N-(5-acenaphthyl)-N'-(4-isopropyl-                                                                68 (±1)                                                                              6.5    ± 1                                   phenyl)guanidine                                                              N-(Adamantan-1-yl)-N'-(adamantan-                                                                 49 (±8)                                                                              --                                              2-yl)guanidine                                                                N,N'-Bis(p-tert.-butylphenyl)                                                                     47 (±7)                                                                              --                                              guanidine                                                                     N-(5-acenaphthyl)-N'-(4-fluoro-                                                                   47 (±4)                                                                              --                                              naphthyl)guanidine                                                            N-(5-acenaphthyl)-N'-(4-hydroxy-                                                                  35 (±7)                                                                              --                                              naphthyl)guanidine                                                            N-(5-acenaphthyl)-N'-(4-methoxy-                                                                  56 (±3)                                                                              --                                              naphthyl)guanidine                                                            N-(Adamantan-2-yl)-N'-(5-                                                                         69 (±3)                                                                              4.8                                             acenaphthyl)guanidine                                                         N,N'-Bis (3-acenaphthyl)guanidine                                                                 36 (±14)                                                                             11                                              N,N',N", N'"-tetracyclohexyl-                                                                     74 (±6)                                                                              3                                               hydrazine-dicarboximidamide                                                   N,N'-Di-(adamantan-2-yl)guanidine                                                                 35 (±12)                                                                             16.1   ± 10                                  N,N'-Bis(1-naphthyl)guanidine                                                                     27 (±5)                                                                              23.6   ± 10                                  N,N'-Di-(4-isopropylphenyl)                                                                       48 (±14)                                                                             19     ± 7                                   guanidine                                                                     N-(5-acenaphthyl)-N'-(adamant-1-                                                                  47 ± 11                                                                              9.7    ± 3                                   yl)-guanidine                                                                 N-(5-acenaphthyl)-N'-(1-naphthyl)                                                                 40 ± 6 16.2   ± 0.4                                 guanidine                                                                     N,N'-Di-(adamantan-1-yl)guanidine                                                                 25 (±11)                                                                             25     ± 9                                   ______________________________________                                    

Table IV shows the percentage inhibition of glutamate release(persistent component) by certain N,N'-disubstituted guanidines of theinvention.

                  TABLE IV                                                        ______________________________________                                        Inhibition of                                                                 Glutamate Release (Persistent Component)                                      By Substituted Guanidines and Related Compounds                                                   Inhibition of                                                                 Glutamate Release                                                               %                                                                             Inhibition                                                                              IC50,                                         Compound              @ 10 uM   uM                                            ______________________________________                                        N,N'-Bis(5-acenaphthyl)guanidine                                                                    92        3                                             N-(5-acenaphthyl)-N'-(4-fluoronaphthyl)                                                             85        <5                                            guanidine                                                                     N-(5-acenaphthyl)-N'-(4-hydroxynaphthyl)                                                            25                                                      guanidine                                                                     N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)                                                            90                                                      guanidine                                                                     N-(Adamantan-2-yl)-N'-(5-acenaphthyl)                                                               63        5                                             guanidine                                                                     N,N',N", N'"-tetracyclohexylhydrazine-                                                              67                                                      dicarboximidamide                                                             N,N'-Di-(adamantan-2-yl)guanidine                                                                   60                                                      N,N'-Bis(1-naphthyl)guanidine                                                                       31                                                      N,N'-Di-(adamantan-1-yl)guanidine                                                                   49                                                      ______________________________________                                    

Example 11 Inhibition of Glutamate Release of Synaptosomes DepolarizedWith Veratridine

Next, the effect of various concentrations ofN,N'-di-(adamantan-2-yl)guanidine on Ca²⁺ release of synaptosomesdepolarized with veratridine was evaluated. As shown in FIG. 5,increasing concentrations of N,N'-di-(adamantan-2-yl)guanidine caused aninhibition of glutamate release in a dose dependent manner.

Example 12 The Effect of N,N'-di(5-acenaphthyl)guanidine on SodiumCurrents in Neuroblastoma Cells

Whole cell sodium currents were elicited in voltage clamped N1E-115neuroblastoma cells, using the stimulus protocol shown on the top ofFIG. 7. Pressure ejection of 30 uM N,N'-di(5-acenaphthyl)guanidine ontothese cells (N=5) blocked inward sodium currents by an average of 40%(49% inhibition in the cell shown, the results for which are depicted inFIG. 7). The effects of this drug were partially reversed followingwashout.

Example 13 Monitoring of the Use-Dependence with the Superfusion System

³ H-glutamate loaded synaptosomes were washed for 10 sec. to washout theexterior radioactivity and the superfusion was started. The superfusionconsisted of 2 sec. low-K⁺ (3 mM) buffer flow followed by 1 sec. ofhigh-K⁺ (55 mM) flow. This was repeated three more timed followed by 2sec. of low-K⁺ buffer. The results are shown in FIGS. 8 and 9.Ca-independent and Ca-dependent glutamate release in the later pulseswere expressed as a percent of their initial fluxes. FIGS. 8 and 9 showthat Ca-independent glutamate release is fairly constant whereas theCa-dependent component decays and requires time to recover.

Example 14 Electrophysiology Studies on N-type Channels

Single bullfrog dorsal root ganglion cells, which expressω-conotoxin-sensitive N-type calcium channels, were treated with 5 uM ofN,N'-di(acenaphthyl) guanidine. The solution also contained (in mM) CsCl90; creatine phosphate, 5; MgATP, 5; tris GTP, 0.3; EGTA, 10; HEPES, 10;pH 7.2. As shown in FIG. 10, this compound inhibited about 40% of thecurrent. At 20 uM, this compound inhibited nearly all of the current(not shown). Most of the inhibition was reversed in about 6 minutes (notshown). Calcium channel rundown during the recording period may accountfor the lack of complete reversal of inhibition. This further supportsthe hypothesis that certain substituted guanidines inhibitneurotransmitter release by clocking presynaptic calcium channels.

EXAMPLE 15 Electrophysiology Studies on Excitatory Synaptic Transmission

Transverse sections of hippocampus were obtained from 4-6 week oldSprague-Dawley rats and maintained under standard conditions. As shownin FIG. 11, synaptic potentials were elicited by application of constantcurrent electrical stimuli delivered through concentric bipolarplatinum/iridium electrodes placed in the stratum radiatum to ensurefull stimulation of the Schaffer collaterals. Extracellular excitatorypost synaptic potentials (EPSP's) were recorded by placing asaline-filled glass microelectrode in either the stratum radiatum orstratum lacunosum. Population spikes were recorded by placing anelectrode in the stratum pyramidole.

As shown in FIG. 12A, the bath application ofN,N'-di(5-acenaphthyl)guanidine completely eliminated population spikes.This effect is completely reversible following washout. The upwardlygoing field EPSP was also reversibly blocked by bath perfusion of thedrug.

As shown in FIG. 12B, the bath application ofN,N'-di(5-acenaphthyl)guanidine almost completely eliminated the fieldEPSP. This effect is fully reversible following washout. Smallpopulation spikes are also evident superimposed on the late phase of theEPSP. The bath perfusion of this compound also reversibly blocked thesepopulation spikes.

FIG. 12C depicts the effect of 20 uM of N,N'-di(5-acenaphthyl)guanidineon the amplitude of the response to an electrical stimulus. EPSPamplitude was reduced between 80-90% and the response was fullyrecovered following washout.

FIG. 13 depicts two graphs showing the effect of 1 mM adenosine (a knownpresynaptic transmitter release blocker) on EPSP amplitude inhippocampal slices. FIG. 13A shows that the bath application ofadenosine almost completely eliminated field EPSP's in a fullyreversible fashion. There was a noticeable overshoot following recovery.Population spikes occurring during the late phase of EPSP were totallyblocked by adenosine but did not appear to return following washout. Thepresence and duration of the presynaptic volley was unaffected byadenosine; however, there appeared to be a small reversible change inamplitude. While transmitter release may be blocked, there is nomeasurable effect on the afferent volley. In addition, this experimentalparadigm cannot distinguish between presynaptically and postsynapticallymediated reductions of extracellularly measured synaptic potentials.

FIG. 13B shows the effect of 1 mM adenosine on normalized EPSP amplitude(measured EPSP amplitude divided by maximum EPSP amplitude inmillivolts) over time. Bath application of adenosine caused a 90%reduction in the EPSP amplitude of the field EPSP (first response topaired stimuli shown here). This effect is fully reversible followingwashout with some overshoot evident in this slice.

The results of the hippocampal slice recordings lead to the followingconclusions. The bath application of N,N'-di(5-acenaphthyl)guanidine (20uM) substantially and reversibly reduced the amplitude of both fieldEPSP's and population spikes by 80-90%. The effects of this compoundwere fully reversible, The effects of this compound qualitativelyresembles the actions of adenosine, an agonist of the presynapticadenosine receptors which is known to block glutamate release. This isconsistent with presynaptic inhibition of neurotransmitter release bythe compound.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method for blocking voltage sensitive calciumchannels of mammalian neuronal cells comprising the administration to amammal in need of such treatment, an effective amount of a compoundhaving the formula: ##STR26## wherein R is cycloalkyl of 3 to 12 carbonatoms, carbocyclic aryl, alkaryl, aralkyl or heterocyclic having 6 to 18carbon atoms and 1-3 separate of fused rings, and 0-5 O, N, and/or ringatoms in an aryl, alicyclic or mixed ring system;R¹ and R² are the sameor different and selected from the group consisting of hydrogen, lowerC₁₋₆ alkyl, lower C₁₋₆ alkylamino, C₅₋₁₀ aryl or substitutedaryl;wherein R and the acenaphthyl group are optionally substituted byhydroxy, acetate, oxo, amino, lower C₁₋₆ alkyl, lower C₁₋₆ alkyl amino,alkoxy of 1-6 carbon atoms, di-lower C₂₋₁₂ alkyl amino, nitro, azido,sulfhydryl, cyano, isocyanato, halogen, amido, sufonato or carbamido. 2.The method of claim 1, wherein said compound is selected from the groupconsisting of N,N'-di-(1-acenaphthyl)guanidine,N,N'-di-(3-acenaphtyl)guanidine, N,N'-di-(5-acenaphthyl)guanidine,N,N'-di-(acenaphthylen-1-yl)guanidine,N-(adamentan-1-yl)-N'-(5-acenaphthyl)guanidine;N-adamantan-2-yl)-N-(5-acenaphthyl)guanidine;N-(adamantan-1-yl)-N-(3-acenaphthyl)guanidine;N-(adamantan-2-yl)-N'-(3-acenaphthyl)guanidine;N-(adamant-1-yl)-N'-(adamant-2-yl)guanidine;N-(3-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N-(4-cyanonaphthyl)guanidine;N-(5-acenaphthyl)-N-(4-cycanonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-amidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-amindonaphthyl)guanidine;N-(3-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(5-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(7-fluroronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(7-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(5-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(3-acenaphthyl)-N'-(4-n-propylphenyl)guanidine;N-(5-acenaphthyl)-N'-(4-n-propylphenyl)guanidine;N-(3-acenaphthyl)-N-(2-isopropylphenyl)guanidine;N-(5-acenaphthyl)-N-(2-isopropylphenyl)guanidine;N-(3-acenaphthyl)-N-(4-cyclorpopylphenyl)guanidine;N-(5-acenaphthyl)-N-(4-cyclorpopylphenyl)guanidine;N-(3-acenaphthyl)-N'-(coumarinyl)guanidine;N-(5-acenaphthyl)-N'-(coumarinyl)guanidine;N-(3-acenaphthyl)-N'-(quinolinyl)guanidine;N-(5-acenaphthyl)-N'-(quinolinyl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N,N'-bis(4-bromo-3-acenaphthyl)guanidine;N,N'(4-bromo-5-acenaphthyl)guanidine;N,N'-bis(4-hydroxy-3-acenaphthyl)-guanidine;N,N'-bis(4-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(4-amino-3-acenaphthyl)guanidine;N,N'-bis(4-amino-5-acenaphthyl)guanidine;N,N'-bis(4-nitro-3-acenaphthyl)guanidine;N,N'-bis(4-nitro-5-acenaphthyl)guanidine;N,N'-bis(1-bromo-3-acenaphthyl)-guanidine;N,N'-bis(1-bromo-5-acenaphthyl)guanidine;N,N'-bis(2-bromo-3-acenaphthyl)guanidine;N,N'-bis(2-bromo-5-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(1-oxo-3-acenaphthyl)guanidine;N,N'-bis(1-oxo-5-acenaphthyl)guanidine;N,N'-bis(2-oxo-5-acenaphthyl)guanidine;N,N'-bis(2-oxo-5-acenaphthyl)guanidine;N,N'-bis(5-acenaphthylenyl)guanidine; N,N¹-N,N'-bis(5-acenaphthylenyl)guanidine;Bis(4-azido-5-acenaphthyl)guanidine; N,N¹-Bis(4-sulfonyl-5-acenaphthyl)guanidine.
 3. The method of claim 1wherein R is optionally substituted cyclopropyl, cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, 1,4-methylenecyclohexyl,1-adamantyl, 2-adamantyl, exo 2-norbornyl, endo 2-norbornyl, exo2-isobornyl, endo 2-isobornyl, menthyl, cyclopentylmethyl,cyclohexylmethyl, 1-cyclohexylethyl, 2-cyclohexylethyl,1-cyclohexylpropyl, 2-cyclohexylpropyl or 3-cyclohexylpropyl.
 4. Themethod of claim 3 where R is optionally substituted carbocyclic aryl,alkaryl, aralkyl or heterocyclic.
 5. The method of claim 1 where R isoptionally substituted phenyl, benzyl, naphthyl, 3-acenaphthyl, or5-acenaphthyl.
 6. The method of claim 1 where R is optionallysubstituted phenyl, benzyl, 1-phenylethyl, 2-phenylethyl,1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, o-tolyl, m-tolyl,p-tolyl, m,m'-dimethylphenyl, o-ethylphenyl, m-ethylphenyl,p-ethylphenyl, m,m'-diethylphenyl, m-methyl-m'-ethylphenyl,o-propylphenyl, 1-naphthyl, 2-naphthyl, biphenyl, indanyl, indenyl,3-acenaphthyl, 5-acenaphthyl, 3-acenaphthylene, 5-acenaphthylene,indolyl, benzthiazole, quinolinyl, isoquinolinyl, pyridyl, pyrimidinyl,pyrazinyl, furanyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, coumarinylor imidazolyl.
 7. The method of claim 1 where the mammal is a human. 8.The method of claim 1 where a pharmaceutical composition comprising thecompound and a pharmaceutically acceptable carrier is administered tothe mammal.
 9. The method of claim 1 wherein the compound is selectedfrom the group consisting ofN-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine,N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine,N-(3-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine andN-(5-acenaphthyl-N'-(2-methoxynaphthyl)guanidine.
 10. The method ofclaim 1 wherein the compound is selected from the group consisting ofN-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine orN-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine.
 11. A method forblocking voltage sensitive calcium channels of mammalian neuronal cellscomprising the administration to a mammal in need of such treatment, aneffective amount of a compound having the formula: ##STR27## wherein Ris cycloalkyl of 3 to 12 carbon atoms, carbocyclic aryl, alkaryl,aralkyl or heterocyclic having 6 to 18 carbon atoms and 1-3 separate offused rings, and 0-5 O, N and/or S ring atoms in an aryl, alicyclic ormixed ring system;R¹ and R² are the same or different and selected fromthe group consisting of hydrogen, lower C₁₋₆ alkyl, lower C₁₋₆alkylamino, C₅₋₁₀ aryl or substituted aryl;wherein R and the acenaphthylgroup are optionally substituted by hydroxy, acetate, oxo, amino, lowerC₁₋₆ alkyl, lower C₁₋₆ alkyl amino, alkoxy of 1-6 carbon atoms, di-lowerC₂₋₁₂ alkyl amino, nitro, azido, sulfhydryl, cyano, isocyanato, halogen,amido, sufonato or carbamido; or a pharmaceutically acceptable saltthereof.
 12. The method of claim 11 where R is optionally substitutedphenyl, benzyl, naphthyl, 3-acenaphthyl, or 5-acenaphthyl.
 13. A methodfor blocking voltage sensitive sodium channels of mammalian neuronalcells comprising the administration to a mammal in need of suchtreatment, an effective amount of a compound having the formula:##STR28## wherein R is cycloalkyl of 3 to 12 carbon atoms, carbocyclicaryl, alkaryl, aralkyl or heterocyclic having 6 to 18 carbon atoms and1-3 separate of fused rings, and 0-5 O, N and/or S ring atoms in anaryl, alicyclic or mixed ring system;R¹ and R² are the same or differentand selected from the group consisting of hydrogen, lower C₁₋₆ alkyl,lower C₁₋₆ alkylamino, C₅₋₁₀ aryl or substituted aryl;wherein R and theacenaphthyl group are optionally substituted by hydroxy, acetate, oxo,amino, lower C₁₋₆ alkyl, lower C₁₋₆ alkyl amino, alkoxy of 1-6 carbonatoms, di-lower C₂₋₁₂ alkyl amino, nitro, azido, sulfhydryl, cyano,isocyanato, halogen, amido, sulfonato or carbamido; or apharmaceutically acceptable salt thereof.
 14. The method of claim 13wherein R is optionally substituted cyclopropyl, cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, 1,4-methylenecyclohexyl,1-adamantyl, 2-adamantyl, exo 2-norbornyl, endo 2-norbornyl, exo2-isobornyl, endo 2-isobornyl, menthyl, cyclopentylmethyl,cyclohexylmethyl, 1-cyclohexylethyl, 2-cyclohexylethyl,1-cyclohexylpropyl, 2-cyclohexylpropyl or 3-cyclohexylpropyl.
 15. Themethod of claim 13 where R is optionally substituted carbocyclic aryl,alkaryl, aralkyl or heterocyclic.
 16. The method of claim 13 where R isoptionally substituted phenyl, benzyl, naphthyl, 3-acenaphthyl, or5-acenaphthyl.
 17. The method of claim 13 where R is optionallysubstituted phenyl, benzyl, 1-phenylethyl, 2-phenylethyl,1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, o-tolyl, m-tolyl,p-tolyl, m,m'-dimethylphenyl, o-ethylphenyl, m-ethylphenyl,p-ethylphenyl, m,m'-diethylphenyl, m-methyl-m'-ethylphenyl,o-propylphenyl, 1-naphthyl, 2-naphthyl, biphenyl, indanyl, indenyl,3-acenaphthyl, 5-acenaphthyl, 3-acenaphthylene, 5-acenaphthylene,indolyl, benzthiazole, quinolinyl, isoquinolinyl, pyridyl, pyrimidinyl,pyrazinyl, furanyl, thienyl, pyrrolyl, thiazolyl, oxazolyl, coumarinylor imidazolyl.
 18. The method of claim 13 where the mammal is a human.19. The method of claim 13 where a pharmaceutical composition comprisingthe compound and a pharmaceutically acceptable carrier is administeredto the mammal.
 20. The method of claim 13 wherein the compound isselected from the group consisting ofN-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine,N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine,N-(3-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine andN-(5-acenaphthyl-N'-(2-methoxynaphthyl)guanidine.
 21. The method ofclaim 13 wherein the compound is selected from the group consisting ofN-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine orN-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine.
 22. The method ofclaim 13 where the compound is selected from the group consistingof:N,N'-di-(1-acenaphthyl)guanidine; N,N'-di-(3-acenaphthyl)guanidine;N,N'-di-(5-acenaphthyl)guanidine; N,N'-di-(acenaphthylen-1-yl)guanidine;N-(adamantan-1-yl)-N'-(5-acenaphthyl)guanidine;N-adamantan-2-yl)-N-(5-acenaphthyl)guanidine;N-(adamantan-1-yl)-N-(3-acenaphthyl)guanidine;N-(adamantan-2-yl)-N'-(3-acenaphthyl)guanidine;N-(adamant-1-yl)-N'-(adamant-2-yl)guanidine;N-(3-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-nitronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-azidonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-bromonaphthyl)guanidine;N-(3-acenaphthyl)-N-(4-cyanonaphthyl)guanidine;N-(5-acenaphthyl)-N-(4-cyanonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-amidonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(4-amidonaphthyl)guanidine;N-(3-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(5-acenaphthyl)-(4-iodonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(7-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(7-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-fluoronaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-methoxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-hydroxynaphthyl)guanidine;N-(3-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(5-acenaphthyl)-N'-(2-aminonaphthyl)guanidine;N-(3-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(5-acenaphthyl)-N'-(4-isopropylphenyl)guanidine;N-(3-acenaphthyl)-N'-(4-n-propylphenyl)guanidine;N-(5-acenaphthyl)-N'-(4-n-propylphenyl)guanidine;N-(3-acenaphthyl)-N-(2-isopropylphenyl)guanidine;N-(5-acenaphthyl)-N-(2-isopropylphenyl)guanidine;N-(3-acenaphthyl)-N-(4-cyclopropylphenyl)guanidine;N-(5-acenaphthyl)-N-(4-cyclopropylphenyl)guanidine;N-(3-acenaphthyl)-N'-(coumarinyl)guanidine;N-(5-acenaphthyl)-N'-(coumarinyl)guanidine;N-(3-acenaphthyl)-N'-(quinolinyl)guanidine;N-(5-acenaphthyl)-N'-(quinolinyl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-nitro-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-amino-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-methoxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(4-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-oxo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-oxo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-bromo-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(1-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-3-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-1-yl)guanidine;N-(2-hydroxy-5-acenaphthyl)-N'-(adamant-2-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(3-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-1-yl)guanidine;N-(5-acenaphthylenyl)-N'-(adamant-2-yl)guanidine;N,N'-bis(4-bromo-3-acenaphthyl)guanidine;N,N'-bis(4-bromo-5-acenaphthyl)guanidine;N,N'-bis(4-hydroxy-3-acenaphthyl)-guanidine;N,N'-bis(4-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(4-amino-3-acenaphthyl)guanidine;N,N'-bis(4-amino-5-acenaphthyl)guanidine;N,N'-bis(4-nitro-3-acenaphthyl)guanidine;N,N'-bis(4-nitro-5-acenaphthyl)guanidine;N,N'-bis(1-bromo-3-acenaphthyl)guanidine;N,N'-bis(1-bromo-5-acenaphthyl)guanidine;N,N'-bis(2-bromo-3-acenaphthyl)guanidine;N,N'-bis(2-bromo-5-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(1-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-3-acenaphthyl)guanidine;N,N'-bis(2-hydroxy-5-acenaphthyl)guanidine;N,N'-bis(1-oxo-3-acenaphthyl)guanidine;N,N'-bis(1-oxo-5-acenaphthyl)guanidine;N,N'-bis(2-oxo-3-acenaphthyl)guanidine;N,N'-bis(2-oxo-5-acenaphthyl)guanidine;N,N'-bis(3-acenaphthylenyl)guanidine;N,N'-bis(5-acenaphthylenyl)guanidine; N,N¹-bis(4-azido-5-acenaphthyl)guanidine; and N,N¹-bis(4-sulfonyl-5-acenaphthyl)guanidine.
 23. The method of claim 13where the mammal is suffering or susceptible to epilepsy.
 24. The methodof claim 21 where the mammal is suffering or susceptible to epilepsy.25. The method of claim 22 where the mammal is suffering or susceptibleto epilepsy.
 26. The method of claim 1 wherein R¹ and R² are eachindependently hydrogen or lower C₁₋₆ alkyl.
 27. The method of claim 1wherein R¹ and R² are each hydrogen.
 28. The method of claim of any oneof claims 1, 11, 13, and 20-25, wherein said compound is administered aspart of a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier.